Screening and use of reagents which block or activate intein splicing utilizing natural or homologous exteins

ABSTRACT

In accordance with the present invention, there are provided selection systems and methods for screening for agents that control splicing of inteins in their native host protein (extein) or in homologous exteins. Specifically, there are provided positive genetic selection systems for the screening of agents which inhibit or activate protein splicing which comprise: a host cell containing a chromosomal gene encoding either a drug-resistant form of a target enzyme or a wild-type target enzyme, and a plasmid-borne gene encoding either a drug-sensitive form of the target enzyme, which is dominantly cytotoxic upon interaction with the drug, or a dominantly cytotoxic form of the target enzyme. In these systems the plasmid-borne gene contains an intein, and the inhibition or activation of splicing of the dominant cytotoxic form of the target enzyme by a given reagent results in the survival or death of the host cell. More specifically, positive genetic selection systems which utilize the  M. xenopi  GyrA intein or  M. tuberculosis  DnaB helicase intein are provided. Similar reporter systems utilizing native or homologous exteins and systems utilizing controllable inteins are provided, as are methods of controlling in vivo expression of proteins by modulating protein splicing with inhibiting or activating agents, and methods of controlling the delivery of proteinaceous drugs in vivo by modulating protein splicing.

RELATED APPLICATIONS

[0001] This Application is a Continuation-In-Part of U.S. Pat. No.5,834,247, issued Nov. 10, 1998, the disclosure of which is herebyincorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to the screening for and use ofagents which either inhibit or activate protein splicing of inteins(IVPS). Specifically, disclosed herein is the development of 2 specificreporter systems for Gyrase A and DnaB inteins. Agents screened for inaccordance with the present invention can be used to control proteinsplicing for any purpose, in vivo or in vitro, including antimicrobialactivity of organisms containing inteins in essential genes. Morespecifically, the present invention relates to the use of inteinsexpressed in modified or unmodified native protein splicing precursorsor homologous extein systems to screen for mutations that modulatesplicing or agents that inhibit or activate splicing. The presentinvention improves on current reporter systems used to screen for agentsthat can control splicing by using a modified or unmodified nativeprecursor or precursor homolog in order to take advantage of the morenative intein active site formed by natural precursors or inteins inhomologous exteins, since agents that are derived from non-nativeprecursors may not have the identical selected activity on nativeprecursors.

[0003] Production of mature proteins involves the flow of informationfrom DNA to RNA to protein. Precise excision of DNA and RNA elementswhich interrupt that information has been previously described (M.Belfort, Annu. Rev. Genet. 24:363 (1990); T. R. Cech, Annu. Rev.Biochem. 59:543 (1990); Hunter et al., Genes Dev. 3:2101 (1989)). Morerecently, evidence for the precise excision of intervening proteinsequences has also been described for the TFPI allele from Saccharomycescerevisiae (Hirata et al., J. Biol. Chem. 265:6726 (1990); Kane et al.,Science 250:651 (1990)) and the recA gene from Mycobacteriumtuberculosis (Davis et al., J. Bact. 173:5653 (1991); Davis et al., Cell71:1 (1992)). Each of these genes contains internal in-frame peptidesegments which must be removed to produce the mature protein. Expressionof Tfp1 and RecA each results in two peptides: one representing theintervening protein sequence (IVPS) and the other the ligated product ofthe external protein sequences (EPS). In 1994, the terms “intein” and“extein” were adopted in place of IVPS and EPS, respectively (Perler, etal., Nucleic Acids Res. 22:1125-1127 (1994)). This post-translationalprocessing event has been termed “protein splicing”. Similarly, the“Vent”® DNA polymerase gene from the hyperthermophilic archaeonThermococcus litoralis contains two in-frame IVPS (Perler, et al., PNAS89:5577 (1992)) and the DNA polymerase gene from the hyperthermophilicarchaeon Pyrococcus species GB-D contains one intein (Xu, M., et al.,Cell 75, 1371-1377 (1993)).

[0004] Over 80 inteins have been identified in bacteria, archaea andeucarya (Perler, F. B., et al. Nucleic Acids Res 25, 1087-93 (1997),Dalgaard, J. Z., et al., J Comput Biol 4, 193-214 (1997), Pietrokovski,S., Protein Sci. 7, 64-71 (1998) and Perler, F. B. Nucleic Acids Res.27, 346-47 (1999). Four inteins have been found in Mycobacterium leprae(Davis, E. O., et al., EMBO J. 13, 699-703 (1994) and Smith, D. R., andet al. Genome Res 7, 802-19 (1997)) and three inteins in Mycobacteriumtuberculosis (Cole, S. T., et al. . Nature 393, 537-44 (1998)). Oneintein has been found in Candida tropicalis (Gu, et al., J. Biol. Chem.,268(10):7372-7381 (1993)).

[0005] Controllable IVPS (CIVPS) and methods for using the same tomodify, produce and purify target proteins has been described (Comb etal., U.S. Pat. No. 5,496,714, issued Mar. 5, 1996; Comb et al., U.S.Pat. No. 5,834,247, issued Nov. 10, 1998). Methods for using inteins toscreen for peptides (or derivative, analogic or mimetic thereof) or anyagent that can enter cells to block or activate splicing of a natural orexperimental reporter protein have also been described (U.S. Pat. No.5,834,247, supra.. at Example 17). These methods specifically describethe screening of peptides using mycobacterial inteins as targets. Thepreparation of an in vivo peptide library utilizing chicken α-spectrinis also described.

[0006] While a general method of screening for antimicrobial agentsusing the M. tuberculosis RecA intein in a thymidylate synthetase (TS)reporter system has been described (Belfort, U.S. Pat. No. 5,795,731,issued Aug. 18, 1998), this system suffers from several limitations.Importantly, several studies of protein splicing in foreign contexts(such as the Belfort system) indicate that intein splicing is moreefficient in the native extein than in foreign exteins (Xu, EMBO J.13:5517-5522 (1994), Xu, EMBO J. 15:5146-5153 (1996), Telenti, J.Bacteriol. 179:6379-6382 (1997), Chong J. Biol. Chem, 273:10567-10577(1998), Liu, FEBS Lett. 408:311-314 (1997), Wu, Biochim. Biophys. Acta1387:422-432 (1998B), Nogami Genetics, 147:73-85 (1997), Kawasaki J.Biol. Chem., 272:15668-15674 (1997), Derbyshire, Proc. Natl. Acad. SciUSA, 94:11466-11471 (1997), Southworth, BioTechniques 27:110-121 (1999),FIG. 7)). For example, the use of foreign exteins yieldstemperature-dependent splicing of the Psp-GBD Pol, Mxe GyrA andSynechocystis DnaB inteins (Xu, EMBO J. 13:5517-5522 (1994), Xu, EMBO J.15:5146-5153 (1996), Telenti, J. Bacteriol. 179:6379-6382 (1997), ChongJ. Biol. Chem, 273:10567-10577 (1998), Liu, FEBS Lett. 408:311-314(1997), Wu, Biochim. Biophys. Acta 1387:422-432 (1998B), NogamiGenetics, 147:73-85 (1997), Kawasaki J. Biol. Chem., 272:15668-15674(1997) and Southworth, BioTechniques, 27:110-121 (1999), and FIG. 7).

[0007] While not wishing to be bound by theory, it is believed that suchinefficient protein splicing in the foreign extein context occursbecause the flanking extein is, in effect, the substrate of the intein.It is, therefore, likely that the intein may exhibit substratespecificity like all other enzymes. The substrate specificity of theintein limits acceptable extein sequences, hence the native exteinsequence is the optimal substrate, whereas foreign extein sequences maynot be acceptable substrates at all. For example, studies of the Sce VMAand Mxe GyrA inteins indicate that thiol induced N-terminal splicejunction cleavage and splicing are, to varying extents, dependent on thesingle extein residue preceding the intein (Chong, J. Biochem.273:10567-10577 (1998), Southworth, BioTechniques, 27:110-121 (1999)).Other extein residues have also been shown to affect splicing of the SceVMA intein (Nogami Genetics, 147:73-85 (1997), Kawasaki J. Biol. Chem.,272:15668-15674 (1997)).

[0008] Additionally, exteins may affect the packing at the intein activesite, or global folding of the intein and/or precursor, hence the use ofa foreign extein may result in improper folding of the intein orprecursor and inefficient or no splicing. Moreover, expression of anextein gene that naturally contains an intein in a foreign host, forexample E. coli or yeast, may not be efficient (Perler et al. Proc.Natl. Acad. Sci. USA 89:5577-5581 (1992) and Hodges, et al., NucleicAcid Res. 20:6153-6157 (1992)), whereas expression of the homologousendogenous extein is likely to be more efficient. For example, theMycobacterium leprae RecA intein fails to splice in E. coli, while itsplices in M. leprae (Davis, et al., EMBO J., 13:699-703 (1994)). It ispossible that the M. leprae RecA intein would splice in E. coli RecA,although that has yet to be tested. In another example, theSynechocystis sp. strain PCC6803 DnaB gene, containing an intein, wasunclonable in E. coli ( Wu, et al., Proc. Natl. Acad. Sci. USA95:9226-9231 (1998)). The M. leprae GyrA precursor did not spliceefficiently in E. coli and was mostly insoluble, while the homologousMxe GyrA intein spliced efficiently in E. coli GyrA.

[0009] Additionally, the use of homologous exteins would eliminate, inmany instances, the need to introduce silent mutations in the reportergene in order to insert the desired intein (see Belfort, supra., Comb,supra, Example 17). Homologous exteins may have naturally-occuring,conserved restriction enzyme sites that would allow the cloning of theintein into the homologous extein or they may have enough exteinsimilarity to allow insertion of the intein into the homologous exteinby recombination. Such systems also eliminate the need for an exogenousreporter gene, since innate extein properties of the native extein maybe used for selection. Alternatively, the native extein may be mutated,either de novo or based on mutations in similar extein genes, to makethe extein into a selectable marker or reporter gene.

[0010] Accordingly, the most desirable intein splicing systems would bethose systems which express an intein from one organism in thehomologous extein from the foreign host organism used for expression orto express the native precursor gene in a suitable foreign hostorganism.

[0011] Such intein systems would not only be useful in the screening ofantimicrobial agents which inhibit intein splicing within a reportergene (as described in Belfort, supra, Comb, supra..), but ascontrollable targets to direct expression of an extein product. Agents,for example peptides, that block intein splicing may be used to limitthe expression of an extein in such systems. The suppression of suchexpression may be highly useful in the drug delivery context, where, forexample, one wishes to turn on an enzyme which is active in killingcancer cells, or by delivering needed activity, for example insulin.

[0012] Similarly, such intein systems may utilize splicing-incompetentinteins to screen for agents with the ability to activate splicing.

SUMMARY OF THE INVENTION

[0013] In accordance with the present invention, there is providedselection systems and methods for selecting or screening for mutationsor agents that control the splicing of inteins which comprise use of theintein's native host protein (extein) or a homologous extein in any hostorganism (FIG. 8). Specifically, in one preferred embodiment, there isprovided a positive genetic selection system for the screening of agentswhich inhibit protein splicing which comprises: a host cell whichcontains (1) a copy of the extein gene (either episomal, chromosomal orsynthetic) gene encoding a mutant or naturally drug-resistant form of atarget enzyme (which as used herein includes not only enzymes, butproteins, peptides or the like), and (2) a wild-type or mutant form ofthe extein gene (either episomal, chromosomal, or synthetic) encoding adrug-sensitive form of the target enzyme which is dominantly cytotoxicupon interaction with the drug, wherein the gene encoding thedrug-sensitive form of the target enzyme contains an intein, and whereinthe inhibition of splicing of the drug-sensitive form of the targetenzyme by a given reagent results in the survival of the host cell inthe presence of the drug. In one specific embodiment, a positive geneticselection system which utilizes the M. xenopi GyrA intein is provided.This system is also applicable to any GyrA intein inserted at the sameor different site in the GyrA extein gene.

[0014] In accordance with another preferred embodiment, there isprovided a similar positive genetic selection system for the screeningof agents which inhibit protein splicing which comprisesa host cellwhich contains (1) a copy of the extein gene (either episomal,chromosomal or synthetic) encoding a wild type form of a target enzyme,and (2) a gene encoding a dominant cytotoxic form of the target enzyme(either episomal, chromosomal or synthetic) wherein the gene encodingthe dominantly and cytotoxic form of the target enzyme contains anintein, and wherein the inhibition of splicing of the cytotoxic form ofthe target enzyme by a given reagent results in the survival of the hostcell. In one particularly preferred embodiment, a positive geneticselection system which utilizes the M. tuberculosis DnaB helicase inteinis provided. This positive selection system may also employ any DnaBintein inserted at the same or different site in the DnaB extein gene.Similar systems and methods of screening for agents that activateprotein splicing are also provided, as are reporter systems utilizingnative or homologous exteins and systems utilizing inteins.

[0015] Also provided by the present invention are methods of controllingin vivo expression of proteins by modulating protein splicing withinhibiting or activating agents. Similar methods of controlling thedelivery of proteinaceous drugs in vivo by modulating protein splicingare also provided.

[0016] As used herein, “agent” includes, but is not limited to, apeptide (free or displayed on a scaffold such as chicken α-spectrin), apeptide derivative, analogic or mimetic, a natural product or asynthetic molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a diagram depicting a protein splicing precursor andproducts and alternative names for each element or part thereof.

[0018]FIG. 2 is a diagram depicting a general scheme for the selectionof peptides which block intein splicing of a dominant lethal suicidegene in vivo.

[0019]FIG. 3A is a diagram depicting the irreversible blocking of DNAreplication by E. coli GyrA interaction with a drug (ofloxacin).

[0020]FIG. 3B is a diagram depicting a Mxe GyrA intein-splicing systemfor the selection of agents which block intein splicing. The splicing ofthe Mxe GyrA intein out of the homologous Eco GyrA extein produces adrug sensitive, wild-type Eco GyrA which, in the presence of ofloxacin,forms an irreversible covalent poison complex during replication thatkills the cell, despite the presence of the chromosomal mutant GyrAwhich is drug resistant.

[0021]FIG. 3C is a diagram depicting a Mxe GyrA intein-splicing systemfor the selection of agents which block intein splicing: the blocking ofsplicing of the Mxe GyrA intein out of the homologous Eco GyrA exteinresults in the expression of the inactive drug sensitive Eco GyrA andthe chromosomal mutant GyrA, which is ofloxacin-resistant. Hence, in theabsence of an active drug sensitive Eco GyrA (due to the blockage ofsplicing,) the host grows.

[0022]FIG. 3D is an amino acid sequence comparison of part of the E.coli GyrA (SEQ ID NO:1) and M. xenopi GyrA (SEQ ID NO:2) sequences,indicating that the GyrA exteins are very similar in amino acidsequence, especially at the intein insertion site marked by the arrow.

[0023]FIG. 3E is a gel indicating efficient splicing of the Mxe GyrAintein in the homologous Eco GyrA extein. The position of Eco GyrA isindicated by the solid black box and the position of the precursorcomprising the Mxe GyrA intein in Eco GyrA is indicated by the black andwhite boxed marker with the white box indicating presence of the intein.

[0024]FIG. 4A-1 is a diagram depicting the intersection of DnaB withDnaC which is required to load DnaB onto the DNA replication machinery.

[0025]FIG. 4A-2 is a diagram depicting the sequestration of DnaC by amutant E. coli DnaB helicase which leads to disrupted DNA replicationand cell death.

[0026]FIG. 4B is a diagram depicting a Mtu DnaB helicase intein-splicingsystem for the selection of agents which block intein splicing: thesplicing of the Mtu DnaB helicase intein out of a dominant lethal mutantMtu DnaB helicase extein produces mutant Mtu DnaB helicase whichsequesters Eco DnaC and poisons replication despite the presence of thechromosomal Eco DnaB helicase, as a result, the host dies.

[0027]FIG. 4C is a diagram depicting a Mtu DnaB helicase intein-splicingsystem for the selection of agents which block intein splicing: theblocking of splicing of Mtu DnaB helicase intein out of a dominantlethal mutant Mtu DnaB helicase extein prevents the sequestration of EcoDnaC; chromosomal Eco DnaB helicase is expressed and the host grows.

[0028]FIG. 4D is an amino acid sequence comparison of part of the E.coli DnaB helicase (SEQ ID NO:3) and M. tuberculosis DnaB helicase (SEQID NO:4) sequences indicating that the amino acid sequences are verysimilar and that the site in E. coli DnaB that was mutated to make itcytotoxic is conserved in M. tuberculosis DnaB (first on largedsequence) and that the intein insertion site is also conserved in E.coli DnaB (marked by the arrow).

[0029]FIG. 5A is a diagram depicting the production of a combinatorialpeptide library using chicken α-spectrin and the screening of thesepeptides for those that block Mxe GyrA helicase intein splicing. “aa”represents a portion of spectrin which can be randomized. The spectrinscaffold is represented by a trapazoid and the different amino acidsequences by various other shapes. If the spectrin binds to theprecursor, splicing is blocked. The system has three components: a hostcell expressing T7 RNA polymerase, the spectrin library and the inteinplus GyrA gene. The latter two genes are present on a single plasmidunder control of T7 RNA polymerase promoters.

[0030]FIG. 5B-1 is a flow diagram indicating multiple-round selection ofcombinatorial peptides that block Mxe GyrA.

[0031]FIG. 5B-2 is a flow diagram indicating Mtu DnaB helicase inteinsplicing.

[0032]FIG. 5C is a gel indicating that peptides p814-818 (SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10)block splicing of Mtu DnaB in E.coli. This is a Western block usinganti-T7 tag antibody to detect the T7 tag at the N-terminus of each DnaBprotein. In p8l5rev, the selected blocking peptide sequence inα-spectrin has been replaced with the wild type spectrin sequence todemonstrate that inhibition of splicing is due to the selected peptidesequence. The bands and markers on the right represent the precursor, aputative C-terminal cleavage product and the spliced DnaB exteins,respectively from top to bottom of the Western Blot. Size markers are inlane M.

[0033]FIG. 6 is a diagram depicting the production of a combinatorialpeptide library using chicken α-spectrin and the screening of thesepeptides for those that block Mtu DnaB intein splicing in Mtu DnaB. E.coli GyrA gyrase. “aa” represents a portion of spectrin which can berandomized. The spectrin scaffold is represented by a trapazoid and thedifferent amino acid sequences by various other shapes. If the spectrinbinds to the precursor, splicing is blocked. The system has threecomponents: a host cell expressing T7 RNA polymerase, the spectrinlibrary and the intein plus GyrA gene. The latter two genes are presenton a single plasmid under control of T7 RNA polymerase promoters.

[0034]FIG. 7 is a table showing the effect of the single amino acidpreceding the Mxe GyrA intein in a heterologous extein context onsplicing and N-terminal cleavage by DTT.

[0035]FIG. 8 is a flow chart for choosing native precursors, homologousexteins or heterologous exteins to develop a selection or reportersystem for testing agents that inhibit or activate splicing of anintein.

[0036]FIG. 9 is a summary of the various methods of selecting agentsthat inhibit or activate protein splicing. Each system is based on amerodiploid cell containing an intein plus and an intein minus exteingene.

[0037]FIG. 10 depicts the scheme for creating random mutations in theMxe gyrA intein by error prone PCR of the intein followed by cloning ofthe mutated intein genes into the E.coli Mxe gyrA extein.

[0038]FIG. 11 depicts the selection scheme based on the GyrA selectiondescribed in Example I in which the presence of a drug kills cells wherethe intein has spliced. Clones that do not splice at Temperature 1 grow,while replica plated clones that splice at lower Temperature 2 do notgrow.

[0039]FIG. 12 show cell lysates from wild type or mutated intein cloneswere electrophoresed in SDS acrylamide gels. A temperature sensitiveclone grown at 37° C. (labeled ‘A’) fails to splice, while the wild typeintein clone splices (labeled ‘WT’). Wild-type levels of splicing areobserved in the mutant clones (1-6) when shifted to 16° C. overnight.

[0040]FIG. 13 illustrates the Mxe GyrA intein sequence (SEQ ID NO:46)with mutations found in the temperature sensitive splicing clonesindicated below the wild-type residue.

[0041]FIG. 14 illustrates the positioning of the mutations in thetemperature sensitive splicing clones on the Mxe GyrA intein 3-Dstructure. The two panels depict opposite sides of the Mxe GyrA inteinwith a single Alanine preceding the intein. Double amino acid changeindicates that the clone had more than one mutation.

DETAILED DESCRIPTION

[0042] The present invention is directed to methods of selecting orscreening for mutations or agents that block or activate proteinsplicing of inteins using natural precursors or by inserting inteins inhomologous extein genes. These mutations or agents can be used toactivate or keep inactive enzymes in vivo or in vitro forpharmacological, chemotherapeutic, or biotechnological purposes. Incontrast, these same methods can be used to select agents that block oractivate splicing in a non-homologous extein if no genetic selectionsystem or screen can be generated for the native extein protein.

[0043] The in vivo control of protein splicing mediated by a blocking oractivating peptide, or other agent that can enter a cell, acting on acontrollable intervening protein sequence (CIVPS) has been described(U.S. Pat. No. 5,834,247, supra. at Example 17). In the presentinvention, it should be noted that a non-controllable IVPS, or intein,is used to identify agents that will convert the IVPS into a CIVPS. Theblocking of such splicing activity by specific agents such as peptidesor natural products, and analogues thereof, is particularly useful incombating pathogens such as Mycobacterium tuberculosis, Mycobacteriumleprae, Mycobacterium avium, or Candida tropicalis by blocking thesplicing of essential proteins in those organisms.

[0044] Approximately 97 inteins have been identified and are availablefrom public databases (Perler, Nucleic Acids Res. 22:1125-1127 (1994),Perler, Nucleic Acids Res. 27:346-347 (1999), Pietrokovski, S., ProteinSci., 7:64-71 (1998) and Dalgaard, et al., J. Comput. Biol., 4:193-214(1997). Sequencing projects of small prokaryotic genomes (e.g.,Mycobacterium tuberculosis, Mycobacterium leprae, and Methanococcusjannaschii) already account for the majority of published inteinsequences. Host genes of these inteins are often involved in suchessential cellular functions as DNA replication, expression, or variousmetabolic processes (compiled in: Perler, Nucleic Acids Res.25:1087-1093 (1997), Perler, Nucleic Acids Res. 27:346-347 (1999),Pietrokovski, S., Protein Sci., 7:64-71 (1998) and Dalgaard, et al., J.Comput. Biol., 4:193-214 (1997). Hence, the disruption of theseessential functions via the blocking of intein splicing by peptides, orother agents, represents a means by which to screen for anti-microbialand anti-pathogenic agents.

[0045] Generally, a positive selection system consists of a gene that isdetrimental to a host organism depending on the growth media or the hoststrain genetic background. The gene product is toxic for the cell,inhibiting growth or killing the host unless the gene product isinactivated. In the context of a protein splicing genetic system, apositive selection system is defined as a system that allows selectionagainst the splicing of an intein inserted in-frame into a host gene(see FIG. 2). If splicing occurs in the precursor protein containing theintein, the cytotoxic host protein will be active and inhibit cellgrowth or kill the cell; if splicing is disrupted the cytotoxic hostprotein will be inactive and allow cell growth. The same descriptionapplies to reporter systems where detection of the host protein isscored, rather than selection for organism viability. Many reportergenes are known, the most common example is the Blue/white screeninvolving β-galactosidase function on X-gal to produce a blue color.

[0046] In accordance with one embodiment of the present invention, thereis provided a positive selection system for identifying agents whichblock or activate protein splicing which comprises a host cell whichcontains (1) a copy of the extein gene (either episomal, chromosomal orsynthetic) encoding a mutant or naturally drug-resistant form of atarget enzyme; and (2) a wild type or mutant form of the extein gene(either episomal, chromosomal or synthetic) encoding the target enzymethat is sensitive to the drug, into which is inserted an intein, whereinthe spliced form of the intein-containing target enzyme is toxic to thehost organism upon interaction with a certain drug. In this system, thehost cell so transformed will express the drug sensitive enzyme if theintein is properly spliced, resulting in reduced viability of theorganism because the spliced product is dominantly lethal or cytotoxicto the host organism, despite the fact that the drug-resistanthomologous gene is also expressed. The intein-les copy of the gene isrequired to maintain viability in cells in which splicing of the plasmidborne extein gene is blocked.

[0047] In one preferred embodiment, a plasmid-encoded, drug sensitivegene is the naturally occurring intein/extein precursor or its homologcontaining an intein, which intein may be either naturally occurring orinserted, and drug sensitivity may be naturally occurring in thisprecursor gene or introduced by mutation in the extein portion of thegene. This system results in death of all cells where splicing occursand thus provides a system for selecting for mutations, drugs,chemicals, peptides, etc. which block splicing in vivo since cellviability requires blockage of splicing.

[0048] In a particularly preferred embodiment, the Mycobacterium xenopiGyrA intein (Mxe GyrA) (SEQ ID NO:1 1) (Telenti et al, J. Bacteriol.179:6378-6382 (1997) is inserted into the homologous extein of E. coliGyrA (see FIG. 3D). In E. coli, GyrA is an essential gene that encodesfor the A subunit of the E. coli gyrase hetero-tetramer protein complex.E. coli gyrase is a type II topoisomerase involved in DNA relaxation atthe origin of replication of the bacterial chromosomal DNA (Swanberg andWang, J. Mol. Biol. 197:729-736 (1987)). The wild type E. coli GyrAbinds irreversibly to quinoline drugs such as ofloaxcin, preventing DNArelaxation during replication, and leading to cell death (see FIG. 3A).However, certain mutants of wild type E. coli GyrA are drug-resistant,while retaining gyrase activity. The generation of an E. coli GyrAmerodiploid host cell which contains a chromosomal copy of adrug-resistant gyrA gene with a second intein-containing, drug sensitivegyrA gene results in a drug sensitive E.coli host, since in this case,drug sensitivity is dominant. The drug sensitive phenotype is dominantbecause the drug sensitive GyrA forms an irreversible covalent poisoncomplex with the drug that interferes with DNA replication (FIG. 3A).

[0049] By merodiploid we mean that the cell contains an extra copy of agene (or several genes) which has been introduced into the cell by anymeans known to one skilled in the art, such as transformation,infection, conjugation, plasmids, virus, phage, or by generating atransgenic strain and which may be present on either an episomal elementor on the host chromosome.

[0050] In accordance with the present invention, there is furtherprovided a similar type of positive selection system (for identifyingagents which block or activate protein splicing) which comprises a hostcell which contains (1) a copy of the extein gene (either episomal,chromosomal or synthetic) encoding a wild type form of a target enzyme,which expresses a non-toxic form of the extein protein; and (2) a seconda extein gene (either episomal, chromosomal or synthetic) encoding acytotoxic form of the target enzyme into which is inserted an intein. Inthis system, the merodiploid host cell expresses the cytotoxic enzyme ifthe intein is properly spliced. Thus, cells must be treated withchemicals, agents or peptides that block splicing of the cytotoxicenzyme at all times when the cytotoxic enzyme is expressed. Thecytotoxic extein must be dominantly lethal, as the intein-less copy ofthe extein gene is also expressed. The intein-less copy of the gene isrequired to maintain viability in cells in which splicing of the plasmidborne extein gene is blocked.

[0051] In one preferred embodiment, instead of using an intein insertedinto a cytotoxic foreign extein homolog, the natural intein precursormay be mutated to produce a cytotoxic extein enzyme after splicing ofthe intein.

[0052] In a particularly preferred embodiment, the Mycobacteriumtuberculosis DnaB precursor (Cole et al., Nature, 393:537-544 (1998)) ismutated in the extein region to a cytotoxic form based on the knowncytotoxic mutation in E.coli DnaB, where Arg231 was mutated to Cysteine(Marszalek and Kaguni, J. Biol. Chem., 267:19334-19340 (1992) andShrimankar, et al., J. Bacteriol., 174:7689-7696 (1992)). DNA helicasesare essential proteins that unwind a DNA duplex to yield asingle-stranded DNA intermediate required for replication,recombination, and repair (LeBowitz and McMacken, J. Biol. Chem.,261:4738-4748 (1986) and Lohman, Mol., Microbiol., 6:5-14 (1992)). Thehexameric E. coli helicase encoded by the dnaB gene interacts with anhexameric DnaC complex and ATP. Some DnaB mutants are dominant lethal(Bouvier and Oreglia, C. R. Acad. Sci. Hebd Seances Acad. Sci. D.,280:649-652 (1975) and Maurer and Wong, J. Bacteriol., 170:3682-3688(1988), Saluja and Godson, J. Bacteriol., 177:1104-1111 (1995) andSclafani, et al., Mol. Gen. Genet. 182:112-118 (1981)). The R231C mutantprotein is deficient in ATP hydrolysis, helicase activity, andreplication activity at the chromosomal origin of replication resultingin cell death. As shown in FIG. 4D, Mtu DnaB contains this same Arginine(R227), and mutating it to Cysteine renders the Mtu DnaB gene cytotoxic.This mutation is dominantly cytotoxic in E. coli, and both the E.coliand M. tuberculosis DnaB proteins result in sequestering of the E. coliDnaC protein into inactive complexes, preventing DnaC from ‘loading’DnaB onto the E. coli DNA replication fork (see FIG. 4A-1 and FIG.4A-2).

[0053] Both of the positive selection systems described in the presentinvention utilize either native or homologous foreign exteins in orderto optimize protein splicing and avoid the ineffecient splicing whichcan result from insertion of the intein into non-homologous foreignextein.

[0054] Co-transformation of the host cell in these positive selectionsystems with a plasmid engineered for the expression of an in vivopeptide library or transformation with a plasmid that contains both theselection marker and the in vivo peptide library (as in FIG. 6) allowsfor the direct selection of clones expressing a peptide that blocks (oralternatively activates) protein splicing. In vivo expression ofpeptides may be hampered by the host's efficient proteolytic degradationsystems. Therefore, expression of these peptides in vivo in the contextof larger proteins is preferred, especially in surface loop regions oflarger proteins. In vivo expression of peptides fused to larger proteinshas been achieved for example, in the catalytic loop of thioredoxin(Colas et al., Nature, 380:548-550 (1996)), and it is possible toexpress peptides fused within many different proteins. Peptidesexpressed in-frame in highly soluble, well expressed, thermostable,solvent-exposed loops of a protein are less subject to in vivoproteolysis or degradation and such fusions enhance the functionalexpression of peptides in a cell.

[0055] In a preferred embodiment, a combinatorial peptide library in afragment of chicken α-spectrin is constructed, as previously described(see U.S. Pat. No. 5,834,247, supra at Example 17). The EF hand regionof chicken α-spectrin was chosen because its structure is known, its EFhand domain forms a small protein with a stable structure, and it has aflexible surface loop. The structure of the chicken α-spectrin EF handdomain was elucidated by NMR analysis (Trave, et al., EMBO J.14:4922-4931 (1995)). The term EF hand describes a type of proteintertiary structural motif consisting of a helix, a turn (loop) and asecond helix. The EF hand domain of chicken α-spectrin is located at thecarboxy terminus of chicken α-spectrin. Its 84 amino acid structure isarranged in two EF hand helix-turn-helix motifs separated by a 14 aminoacid long flexible linker (SEQ ID NO:12). The protein is extremelysoluble without any detectable precipitation or aggregation even atconcentrations of up to 10 mM. The linker loop is mainly unstructured insolution and mutagenesis data show that minor deletions or insertions inthe loop do not disturb the stabilizing hydrophobic interactions betweenthe 2 EF-hand.

[0056] We have taken advantage of this last property to insert randompeptides in the linker region between the chicken α-spectrin EF hands.Peptide libraries of various sizes can be investigated. It will bereadily apparent to the skilled artisan that alternative methods ofproducing in vivo peptide libraries for screening may be utilized andare within the scope of the present invention.

[0057] Although the systems discussed above select for agents that blocksplicing of native or homologous exteins, it will be recognized by thoseof skill in the art that similar strategies can be used for screeningwith reporter genes to look for agents that inhibit expression of activereporter genes. It will likewise be recognized by the skilled artisanthat similar strategies can be used to look for agents that activatesplicing of a splicing-deficient intein in its native context or in ahomologous extein gene, as long as the extein gene can be converted intoa reporter. For example, in one embodiment, a reagent that activates asplicing-deficient precursor results in expression of an active reporterprotein, resulting in inhibition of cell growth or detection of theactive reporter protein.

[0058] Although the Escherichia coli (E. coli) GyrA and theMycobacterium tuberculosis (M. tuberculosis) DnaB selection systems aredescribed above, it will be recognized by the skilled artisan that anygenetic selection system can also be used to isolate peptide sequencesor other agents which disrupt or catalyze protein splicing. Likewise,although we describe the specific use of the M. xenopi GyrA intein (SEQID NO:11) and the M. tuberculosis DnaB intein (SEQ ID NO:13), theskilled artisan will recognize that this strategy is equally applicableto any intein (see, e.g., Perler, et al., Nucleic Acids Res. 27:346-3471999)) present in its native or homologous context. It will likewise bereadily apparent to those of skill in the art that alternative means ofgenerating peptide libraries for screening may be used. It will likewisebe recognized by the skilled artisan that in the absence of a selectionor screening system for the native extein protein that similarstrategies can be applied to splicing of inteins in less optimalheterologous extein systems.

[0059] Activating and/or inhibiting agents identified by the screeningmethods of the instant invention may also be used to control the in vivoexpression of a target protein. Once the only copy of an active exteingene contains an intein, gene function can be inhibited if the organismis treated with an agent that blocks splicing. On the other hand, if asplicing-impaired intein is used, gene function can be activated if theorganism is treated with an agent that activates splicing. The agentsand splicing can be modulated at any time during the development andlife of the organism by addition or removal of the splicing activatingor inhibiting agent.

[0060] Similarly, controllable splicing may be used to deliver activeproteins at specific times or to specific places. In many instances,therapeutic drugs can be cytotoxic to the host and would be best if onlyactive at the target site. For example, chemotherapy drugs are oftengenerally cytotoxic and adverse reactions in normal cells could beeliminated if the drug could be specifically activated in the tumor. Ifone has a drug that is at least partially proteinacious, an intein thatcan be activated or inhibited by a second agent, as described above,could be inserted into the protein portion of the therapeutic agent. Thedrug is then administered in an inactive form, and subsequentlyactivated in the desired target tissue.

[0061] As noted above, it is believed that inefficient protein splicingin the foreign extein context occurs as inteins may exhibit substratespecificity, preferring their native extein sequence to that of foreignexteins. In accordance with another embodiment, there is provided amethod of overcoming this limitation by employing an intein with one ormore, preferably one to five, amino acid residues from its nativeextein. Inclusion of such amino acid residues may be at either or bothends of the homologous intein. Inclusion of amino acid residues from thenative extein will facilitate methodologies of the present invention.Such amino acid residues from the native extein may be incorporated intothe precursor by methods well known to those skilled in the art.

[0062] Insertion of a target intein in the heterologous extein may be atany of a number of sites, including but not limited to, a surfacelocation in the extein, within a loop region of the extein, at aprotease sensitive site, or a position known to facilitate insertion ofadditional amino acid residues without inactivating the extein.

[0063] The Examples presented below are only intended as specificpreferred embodiments of the present invention and are not intended tolimit the scope of the invention except as provided in the claimsherein. The present invention encompasses modifications and variationsof the methods taught herein which would be obvious to one of ordinaryskill in the art.

[0064] The references cited above and below are hereby incorporated byreference herein.

EXAMPLE I A Mxe GyrA Intein-Mediated Positive Selection System forInhibition of Protein Splicing

[0065] A Positive Selection System Based on Blocking Splicing of the MxeGyrA Intein (IVPS) in E. coli GyrA: Background

[0066] Gyrases are essential multimeric enzymes involved in DNAreplication in bacteria (Swanberg and Wang, 1987). Both gyrase subunit Aand B have been extensively studied as drug targets in bacterial humanpathogens (e.g., Mycobacteria, Salmonella, Enterbacteriaceae,Citrobacter, Pseudomonas, Streptococcus, Staphylococcus, Yersinia,Rhodobacter, Haemophilus, Neisseria, Providencia). The GyrA subunit ofgyrases can complex with quinoline drugs, such as ofloxacin, and inducecell death. The complex formation of quinolines with gyrase is followedby a rapid and irreversible inhibition of DNA synthesis, inhibition ofgrowth, and induction of the SOS response (see FIG. 3A). At higher drugconcentrations, cell death occurs as double-strand DNA breaks in thebacterial chromosome are released from trapped gyrase complexes.

[0067] In many gram-negative bacteria (e.g., E. coli), resistance toquinoline arises from mutation of the Gyrase A protein in the quinolineresistance determining region such as gyrA96 or gyrA83 . Those mutationsmay involve Ser83 in E. coli GyrA. In a merodiploid cell containing adrug resistant gyrA gene (such as gyrA83) on the chromosome and a wildtype (gyrA+) copy of gyrA on a plasmid, the wild type gene (drugsensitive) product of gyrA is dominant. By merodiploid, we mean that thecell contains an extra copy of a gene (or several genes) which has beenintroduced into the cell by any means known to one skilled in the art,such as transformation, infection, conjugation, plasmids, virus, phage,or by generating a transgenic strain and which may be present on eitheran episomal element or on the host chromosome. The wild type gyrA genecan be introduced into the cell by any means known to one skilled in theart and should not be considered limited to a plasmid. Many E. colistrains are available that contain gyrA mutants which are resistant toquinoline drugs such as ofloxacin. However, this system is alsoapplicable to any other host system where (1) the chromosomal copy ofthe gyrA gene is resistant to quinoline drugs, (2) the introducedsensitive gyrA gene is present as the heterologous E. coli gyrA::MxegyrA intein fusion or the native Mxe gyrA or M. leprae gyrA genes, and(3) the intein containing drug sensitive gyrA gene is operably linkedwith the appropriate signals for expression in that host. Likewise, theMxe GyrA intein could be inserted into the gyrA gene of any experimentalhost cell just as it was inserted into the E. coli gyrA gene. Likewise,any gyrA intein can be used in the above selection system, whetherpresent at the same site as the Mxe GyrA intein or a different site.

[0068] Since sensitivity to quinoline drugs is dominantly cytotoxic, inthe presence of these drugs, a gyrA+/gyrA83 host cell is not viablebecause wild type GyrA proteins can still bind drug molecules and poisonDNA replication (see FIG. 3B). The co-expression of a chicken α-spectrinpeptide library (as described in U.S. Pat. No. 5,834,247 supra. atExample 17) allows for the positive selection of peptides that candisrupt splicing of the Mxe GyrA intein. Likewise, this system can beused to screen for any agent that inhibits splicing of the Mxe GyrAintein in vivo or for Mxe GyrA intein mutations that block splicing.

[0069] Insertion of the Mxe GyrA intein (IVPS) gene into the homologoussite in the E. coli GyrA gene

[0070] In some Mycobacteria, the gyrase A subunit active site is ofteninterrupted by a naturally occurring allelic intein (IVPS) near theactive site tyrosine residue (e.g., Mycobacterium flavescens,Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium leprae,Mycobacterium malmoense and Mycobacterium xenopi, (Sander, et al.,Microbiology, 144:589-591 (1998), Telenti, et al., J. Bacteriol.,179:6378-6382 (1997), Perler, et al., Nucleic Acids Res. 27:346-347(1999), Southworth, BioTechniques, 27:110-121 (1999)). The M. xenopi(Mxe) GyrA intein (IVPS) utilized in the system described herein, lacksthe endonuclease signature motifs and other sequences similar to homingendonucleases and has been extensively studied as a prototype minimalintein (IVPS) (Kiabunde, et al., Nature Struct. Biol. 5:31-36 (1998),and Telenti, et al., J. Bacteriol., 179:6378-6382 (1997) and SouthworthBioTechniques, 27:110-121 (1999)). The most favorable insertion site forthe Mxe GyrA intein in E. coli GyrA is the homologous insertion sitecompared to the native Mxe GyrA extein, since it shares sequenceidentity with the native Mxe GyrA extein (FIG. 3D). The intein (IVPS)insertion site was chosen immediately upstream of the conserved tyrosineactive site residue at position 122 in E. coli GyrA:

[0071] D S A A A M R Y122 c i t - - - s h n T123 E I R L A K I (SEQ IDNO:14) (SEQ ID NO:45)

[0072] Amino acid numbers refer to the position of the amino acids in E.coli GyrA (see SEQ ID NO:1 for a partial E. coli GyrA sequence). Theunderlined amino acids (single letter amino acid code) are the aminoacids identical in both E. coli and Mxe GyrA exteins (see FIG. 3D). Thelower case letters represent Mxe GyrA intein (IVPS) amino acids. Thedashes represent the remainder of the residues of the Mxe GyrA intein(IVPS) that are not listed (see SEQ ID NO:2, for the complete Mxe GyrAintein sequence).

[0073] First, the E. coli gyrA gene was cloned by polymerase chainreaction (PCR) using E. coli K12 genomic DNA under the followingexperimental conditions. A forward primer 5′-GATAGGCTAGCGATGAGCGACCTTGCGAGAG-3′(SEQ ID NO:15) and reverse primer5′-TGAAGCAATTGAATTATTCTTCTTCTGGCTCG-3′ (SEQ ID NO:16) were used in a PCRmixture containing 20 U/ml Vent® Exo+ DNA polymerase (New EnglandBiolabs, Inc., Beverly, Mass.), 400 μM of each dNTP, 4 nM each primerand 100 ng of E. coli K12 genomic DNA. Amplification was carried out ina Perkin-Elmer/Cetus (Emeryville, Calif.) thermal cycler 480 for 1 minat 95° C. and then cycled at 45° C., 30 sec; 72° C., 2 min and 30 sec;95° C., 30 sec for 20 cycles. The PCR products from one 50 μl PCRreaction and 2 μg of pCYB1 (IMPACT™ I kit, New England Biolabs, Inc.,Beverly, Mass.) were separately digested with 250 U/ml of Nhel and 1000U/ml of Mfel in the presence of 100 μg/ml of BSA. The digestion wasperformed at 37° C. for 1 hour. Digested PCR products and plasmid DNAwere separated by agarose gel electrophoresis and the excised bandsfurther purified using QIAEX II beads as described by the manufacturer(Qiagen, Studio City, Calif.). Ligation was carried out at 20° C. for 1hour using a 1:4 ratio of vector to insert and 40,000 U/ml of T4 ligase.Ligation products were transformed into E. coli strain ER2267 competentcells. Recombinant plasmids were checked by Nhel/Mfel digestion whichresults in the excision of the cloned insert in properly ligatedrecombinants. One of the resultant correct plasmids containing the E.coli gyrA gene placed under transcriptional control of the pCYB1 pTacpromoter was named pEA500. The gyrA insert was checked by DNA sequencingto insure that no sequence errors were introduced by PCR.

[0074] Second, to facilitate cloning of the Mxe GyrA intein into E. coliGyrA, unique silent Notl and Xbal restriction enzyme sites wereengineered 7 bp and 44 bp, respectively, away from each side of the E.coli GyrA active site residue, Y122 of pEA500 by site-directed silentmutagenesis. The QuickChange kit was used following the manufacturer'sinstructions (Stratagene, La Jolla, Calif.) with mutagenic primers: Notloligonucleotides: 5′-CGGCGACTCTGCGGCCGCAATGCGTTATA CGG-3′ (SEQ ID NO:17)and 5′-CCGTATAACGCATTGCGGCCGCA GAGTCGCCG-3′ (SEQ ID NO:18), and Xbaloligonucleotides: 5′-GMCTGATGGCCGCTCTAGAAAAAGA GACGG-3′ (SEQ ID NO:19)and 5′-CCGTCTCTTTTTCTAGAGCGGCCA TCAGTTC-3′(SEQ ID NO:20). The resultantplasmid containing E. coli GyrA with Noti and Xbal restriction enzymesites was called pEA502.

[0075] Third, a 68 bp DNA cassette with flanking Notl/Xbal restrictionsites was designed to be cloned into the pEA502 unique Notl/Xbal sites.This cassette introduced a unique Blpl silent restriction enzyme site 10bp away from Y122 which subsequently allowed cloning of any intein (IVPSor CIVPS) near the E. coli GyrA active site Y122 using Notl and Blplrestriction enzyme sites. This cassette was generated by annealing 2complementary oligonucleotides : 5′-GGCCGCAATGCGTTATACGGAAATCCGCTTAGCGAAAATTGCCCATGMCTGATG GCCGAT-3′ (SEQ ID NO:21)and 5′-CTAGATCGGCATCAGTTCATG GGCAATTTTCGCTAAGCGGATTTCCGTATAACGCATTGC-3′(SEQ ID NO:22). 5 nM of each oligonucleotide was combined in 1×T4 ligasebuffer (New England Biolabs, Inc., Beverly, Mass.), boiled for 5 min andcooled down to room temperature. 10 μg of pEA502 were digested with Notland Xbal using 500 U/ml each enzyme in the presence of 100 μg/ml of BSA.The digestion was performed at 37° C. for 2 hours. The digested plasmidDNA was separated by agarose gel electrophoresis and the excised bandfurther purified using QIAEX II beads as described by the manufacturer(Qiagen, Studio City, Calif.). Ligation of the oligonucleotide cassetteand the digested plasmid DNA was carried out at 20° C. for 1 hour usinga 1:2 ratio of vector to insert and 40,000 U/ml of T4 ligase. Ligationproducts were transformed into E. coli strain ER2267 competent cells.Recombinant plasmids were checked by Blpl digestion which results in thelinearization of the correct recombinant plasmids. One of the resultantcorrect plasmids was named pEA523.

[0076] Fourth, the Mxe GyrA intein (IVPS) was amplified by PCR with theaddition of primer derived Notl and Blpl sites using pMIP(Mxe)#4 plasmidDNA (Telenti et al., J. Bacteriol, 179:6378-6382 (1997)) under thefollowing experimental conditions: Forward primer5′-CGACCCGCGCGGCCGCAATGC GTTATTGCATCACGGGAG-3′ (SEQ ID NO:23) andreverse primer 5′-GCCAAAGGCGCTAAGCGGATTTCCGTGTTGTGGCTGACGMCC CG-3′ (SEQID NO:24) were used in a PCR mixture containing 10 U/ml Taq DNApolymerase (Promega, Madison, Wis.), 200 μM of each dNTP, 4 nM eachprimer and 100 ng pMIP(Mxe)#4 DNA. Amplification was carried out in aPerkin-Elmer/Cetus (Emeryville, Calif.) thermal cycler 480 at 94° C., 30sec; 50° C., 30 sec; 72° C., 15 sec for 10 cycles. The PCR products ofone 50 μl reaction and 2 μg of pEA523 were separately digested using1000 U/ml of Notl and 300 U/ml of Blpl in the presence of 100 μg/ml ofBSA. The digestion was performed at 37° C. for 2 hours. Digested PCRproducts and plasmid DNA were separated by agarose gel electrophoresisand the excised bands further purified using QIAEX II beads as describedby the manufacturer (Qiagen, Studio City, Calif.). Ligation was carriedout at 20° C. for 2 hours using a 1:3 ratio of vector to insert and40,000 U/ml of T4 ligase (New England Biolabs, Inc., Beverly, Mass.).Ligation products were transformed into E. coli strain ToploF′(Invitrogen, Carlsbad, Calif.) competent cells. Recombinant plasmidswere checked by EcoNl restriction enzyme digestion which results in thelinearization of the correct recombinant plasmids. One of the resultantcorrect plasmids was named pEA600 and contains the in-frame insertion ofthe Mxe GyrA intein (IVPS) into the E. coli GyrA extein at the activesite Y122 (see FIGS. 3D and 3E). Construction of a vector forco-expression of a peptide library and the Mxe GyrA intein (IVPS)::E.coli GyrA selection system As described above, the Mxe GyrA intein(IVPS) was inserted into the active site of E. coli GyrA. In thishomologous context, the Mxe GyrA intein (IVPS) splices efficiently toproduce active E. coli GyrA. As is detailed below, the E. coli gyrA::MxegyrA intein (IVPS) gene fusion described above was cloned under controlof a T7 RNA polymerase promoter and introduced into E. coli, ER2726 (NewEngland Biolabs, Inc., Beverly, Mass.). ER2726 expresses T7 RNApolymerase and has the gyrA83 mutation which makes the chromosomal gyrAgene resistant to quinoline drugs. In the presence of quinoline drugssuch as ofloxacin, only splicing deficient clones can survive (see FIGS.3B and 3C), since the spliced gyrA product is sensitive to ofloxacin ina dominant cytotoxic manner (see above).

[0077] The spectrin scaffold was cloned into EA600 as follows. First, a30 bp DNA cassette with flanking PflMI/Apal restriction sites wasdesigned to be cloned into the unique PflMI/Apal sites in pEA600 (whichalso contains the E. coli gyrA::Mxe gyrA fusion). This cassetteintroduced a unique Sphl site in place of the laclq gene and wassynthesized by annealing 2 oligonucleotides: 5′-ATGGGCATGCATATATATATAGGCCTGGGCC-3′ (SEQ ID NO:25) and 5′-CAGGCCTATATATAT ATGCATGCCCATTCG-3′(SEQ ID NO:26). 5 nM of each oligonucleotide was combined in 1×T4 ligasebuffer (New England Biolabs, Inc. Beverly, Mass.), boiled for 5 min andcooled down to room temperature. 5 μg of pEA600 was digested using 320U/ml of PflMI and 800 U/ml of Apal in the presence of 100 μg/ml of BSA.The digestion was performed at 37° C. for 2 hours. The digested plasmidDNA was separated by agarose gel electrophoresis and the excised bandfurther purified using QIAEX II beads as described by the manufacturer(Qiagen, Studio City, Calif.). Ligation was carried out at 16° C. for 1hour using a 1:1 ratio of vector to insert and 40,000 U/ml of T4 ligase.Ligation products were transformed into E. coli strain XL1B (Stratagene,La Jolla, Calif.) competent cells. Recombinant plasmids were checked bySphl digestion which results in the linearization of the correctrecombinant plasmids. One of the resultant correct plasmids was namedpEA661.

[0078] Second, unique Sgfl and sites Clal were engineered on either sideof the spectrin loop region in a spectrin encoding plasmid (Trave, etal., EMBO J. 14:4922-4931 (1995)) by site-directed silent mutagenesisusing the QuickChange kit as described by the manufacturer (Stratagene,La Jolla, Calif.). The Sgfl oligonucleotides were:5′-GTTTAAGTCTTGCTTGCGATC GCTTGGCTATGACCTGCC-3′ (SEQ ID NO:27) and5′-GGGCAGGT CATAGCCAAGCGATCG CAAGCAAGACTTAAA-3′ (SEQ ID NO:28) and Claloligonucleotides were: 5′-GCCTGACCCCGAATTTGAATC GATTCTTGACACTGTTG-3′(SEQ ID NO:29) and 5′-CAACAGTGT CMGAATCGATTCM ATTCGGGGTCAGGC-3′ (SEQ IDNO:30). The resulting plasmid was called pEA670.

[0079] Third, the Sgfl/Clal mutated spectrin gene was cloned by PCR intopEA661 under the following experimental conditions. A forward primer5′-AATGGTGCATGCAAGGAGATGGCGCCCAAC AGTC-3′ (SEQ ID NO:31) and reverseprimer 5′-GCTTTGGCTAG CTTTCCTGTGTCACCTGCTGATCATGMCG-3′ (SEQ ID NO:32)were used as described in the Expand High Fidelity PCR system(Boehringer Mannheim, Indianapolis, Ind.) in the presence of 1×buffer 2(New England Biolabs, Beverly, Mass.) and 50 ng of pEA670 DNA.Amplification was carried out in a Perkin-Elmer/Cetus (Emeryville,Calif.) thermal cycler 480, 94° C., 30 sec; 45° C., 30 sec; 72° C., 45sec; for 15 cycles. The PCR products of one 50 μl tube and 5 μg ofpEA661 were Nhel/Sphl digested using 250 U/ml of each enzyme. Thedigestion was performed at 37° C. for 2 hours. Digested PCR products andplasmid DNA were separated by agarose gel electrophoresis and theexcised bands further purified using QIAEX II beads as described by themanufacturer (Qiagen, Studio City, Calif.). Ligation was carried out at16° C. for 1 hour using a 1:5 ratio of vector to insert and 40,000 U/mlof T4 ligase (New England Biolabs, Inc., Beverly, Mass.). Ligationproducts were transformed into E. coli strain Novablue DE3 (Novagen,Madison, Wis.) competent cells. Recombinant plasmids were checked byNhel/Sphl digestion which results in the excision of the cloned insert.One of the resultant correct plasmids was named pEA671.

[0080] Fourth, the first E. coli gyrA Pvul site in pEA671 was eliminatedby site-directed silent mutagenesis using the QuickChange kit asdescribed by the manufacturer (Stratagene, La Jolla, Calif.) andoligonucleotides 5′-GCGTAAAGCTCGCGACC GTGCTCATATCC-3′ (SEQ ID NO:33) and5′-GGATATGAGCACGGTC GCGAGCTTTACGC-3′ (SEQ ID NO:34), resulting inplasmid pEA681.

[0081] Fifth, the 200 bp Acc651/HindIII fragment from pEA681 wastransferred to pEA671 replacing the Acc691/HindIII fragment of EA671.Plasmids pEA671 and pEA681 were digested in 1×buffer 2 (New EnglandBiolabs, Inc., Beverly, Mass.) using 500 U/ml of Acc651 and 500 U/ml ofHindIII (New England Biolabs, Inc., Beverly, Mass.). The digestion wasperformed at 37° C. for 2 hours. Digested plasmids were separated byagarose gel electrophoresis and the excised bands further purified usingQIAEX II beads as described by the manufacturer (Qiagen, Studio City,Calif.). Ligation was carried out at 16° C. for 3 hours using a 1:3ratio of vector to insert and 40,000 U/ml of T4 ligase (New EnglandBiolabs, Inc., Beverly, Mass.). Ligation products were transformed intoE. coli strain XL1B (Stratagene, La Jolla, Calif.) competent cells.Recombinant plasmids were checked by Pvul digestion. One of theresultant correct plasmids was named pEA682. This plasmid contains boththe α-spectrin peptide library and the E. coli gyrA::Mxe gyrAintein-based selection system on the same plasmid, both under control ofa T7 RNA polymerase promoter (FIG. 5A).

[0082] A Theoretical Screening for Peptides That Disrupt ProteinSplicing of the Mxe GyrA intein (IVPS) in E. coli GyrA.

[0083] In the theoretical embodiment detailed below, random peptides of7-12 amino acids would be inserted in-frame into the loop between the 2EF-hand motifs of α-spectrin, contained, as described above, on the sameplasmid as E. coli gyrA::Mxe gyrA intein fusion. The resulting plasmidswould be electro-transformed into strain ER2726 (New England Biolabs,Inc., Beverly, Mass.). Transformants would be selected in LB liquidgrowth media in the presence of ampicillin, ofloxacin (Sigma, St. Louis,Mo.) and IPTG to allow selection against the splicing proficient clones.Ampicillin selects for the presence of the plasmid and ofloxacin selectsfor peptides that block splicing, since the spliced E. coli GyrA proteinwould be sensitive to the drug and lead to cell death. Plasmid DNA wouldbe isolated from selected clones and digested with Sgfl and Clal toisolate DNA fragments encoding the selected spectrin peptides. Thespectrin DNA loop fragments would then be cloned back into the originalselection plasmid. Iterative rounds of drug selection and “back-cloning”would be performed (FIG. 5B). Iterative screening helps enrich foragents that truly block splicing while eliminating clones that survivedselection because of some other mutation or anomaly. Final selectedclones would be grown individually in liquid culture and theplasmid-encoded E. coli GyrA specifically induced by IPTG. Crude proteincell extracts would be electrophoresed and blotted for immuno-staining.Clones in which the E. coli GyrA spliced product was not detected wouldbe considered positives, i.e. clones in which splicing had beendisrupted, potentially by the selected peptide.

[0084] The random peptide library would be synthesized in vitro usingthe following protocol, as was done in Example II. A singlestrand/double strand DNA hybrid cassette would be synthesized byannealing 2 oligonucleotides: 5′-TGTCAAGAATCGATTCAAATTCGGGGTCAGGCTCTCC((W)NN)₇₋₁₂ATAGCCAAGCGA T-3′ (SEQ ID NO:35)and 5′P-CGCTTGGCTAT-3′ (SEQ ID NO:36). 5 μg of oligonucleotide SEQ IDNO:35 and 3 molar equivalents of oligonucleotide SEQ ID NO:36 would bemixed together in the presence of 0.1 M NaCl in a final volume of 50 μl.The mixture would then be boiled and immediately cool down to roomtemperature in the same boiler. The single stranded random nucleotidepart of the DNA hybrid cassette formed by annealing of the 2 oligoswould be extended using 400 μM of each dNTPs and 60 U/ml of Klenow DNApolymerase (New England Biolabs, Inc., Beverly, Mass.) in a final volumeof 200 μl in 1×EcoPol buffer (New England Biolabs, Beverly, Mass.). Theextension reaction would be left 20 minutes at 37° C. and furtherpurified using QIAEX II beads as described by the manufacturer (Qiagen,Studio City, Calif.). 60 μg of pEA682 (50 μg/ml) would be digested in1×Buffer 2 (New England Biolabs, Inc., Beverly, Mass.) using 250 U/ml ofSgfl (Promega, Madison, Wis.) and 500 U/ml of BspDl (New EnglandBiolabs, Inc., Beverly, Mass.) (an isoschizomer of Clal) in the presenceof 100 μg/ml of BSA. The digestion would be performed at 37° C. for 2hours. Purified cassettes (50 μl) would then be digested in 1×Buffer 4(New England Biolabs, Inc., Beverly, Mass.) using 500 U/ml of Clal (NewEngland Biolabs, Inc., Beverly, Mass.) in the presence of 100 μg/ml ofBSA. Cassettes would be further purified using QIAEX II beads asdescribed by the manufacturer (Qiagen, Studio City, Calif.). Digestedplasmid DNA would be electrophoresed on 0.7% agarose gel and the excisedbands further purified using QIAEX II beads as described by themanufacturer (Qiagen, Studio City, Calif.). Ligation would be carriedout at 16° C. for 1 hour using a 1:1 ratio of vector (2 ng/μl) to insertand 1,600 U/ml of T4 ligase (New England Biolabs, Inc., Beverly, Mass.).Ligation products would then be electro-transformed into E. coli strainER2744 (New England Biolabs, Inc., Beverly, Mass.) competent cells (10⁹pUC18 transformants/μg) using 1-2 μg of total ligated plasmid for each200 μl aliquot of competent cells, at 2.5 kV/cm in a 2 mm cuvette(BIORAD, Richmond, Calif.). Cells would be allowed to recover in ashaker for 1 hour at 37° C. Recovered transformants would be inoculatedat 1/100 dilution ratio into LB liquid growth media containingappropriate amounts of ofloxacin (Sigma, St. Louis, Mo.), 100 μg/ml ofampicillin and 1 mM IPTG. Transformants would be incubated overnight at37° C. Plasmid DNA would be isolated from a 100 ml of the overnightculture using a tip100 column (QIAGEN, Studio City, Calif.), Clal/Sgfldigested as above and electrophoresed on a 4% GTG Nusieve agarose gel(FMC BioProducts, Rockland, Me.). The 57 to 72 bp spectrin loop DNAinserts (depending upon whether the peptide library contained 7 or 12random amino acids) would be purified using QIAEX II beads as describedby the manufacturer (Qiagen, Studio City, Calif.) and cloned back intoSgfl/Clal digested and purified selection plasmid as described above.This protocol would be repeated 3 times to enrich the pool oftransformants for peptide clones having the most biologically activesequences against the protein splicing of the Mxe intein (IVPS). Finallyselected clones would be grown individually in 10 ml LB containing 100μg/ml ampicillin at 37° C. and induced with 1 mM IPTG for 3 hours. Crudeprotein cell extracts would be electrophoresed on a 10-20% gradient gel(Novex, San Diego, Calif.). The gel would then be electro-blotted forimmuno-staining using anti-His tag antibodies (Sigma, St. Louis, Mo.) todetect GyrA::Mxe intein (IVPS) protein splicing products. One wouldexpect to see the absence of spliced product. The clones would then besequenced to determine the amino acid sequences which had been selected.

[0085] Hypothetical Screening with Agents that Inhibit Splicing

[0086] At this stage, the vector, pEA600, is amenable for screening withany type of agent that blocks splicing, using a similar screeningprotocol as for peptides that block splicing, described above. However,in this case, pEA600 or similar plasmids can be directly screenedwithout having to clone the peptide library contained within the chickenα-spectrin gene as described above. The protocol would involve treatingindividual cultures with single or pooled agents that can enter the celland looking for cell growth, using any means known to one skilled in theart. Agents that block splicing allow the cell to grow in the presenceof ofloxacin

SUMMARY

[0087] In summary, we describe the cloning of the Mxe gyrA intein geneinto the E. coli gyrA extein gene for use in selecting for agents thatinhibit splicing. The Mxe GyrA intein splices well in the E. coli GyrAextein, resulting in production of active E. coli GyrA protein. The E.coli GyrA extein was used with the Mxe GyrA intein because the Mle GyrAintein did not splice efficiently in E. coli in its native context andthe precursor was mostly insoluble in E. coli. Because the GyrA inteinand extein sequences are very similar (Telenti, et al., J. bacteriol,179:6378-6382 (1997) and Perler, et al., Nucleic Acids Res. 27:346-347(1999)), mixing and matching of inteins, exteins and experimental hostsresulted in an efficient model system for examining agents that modulatesplicing of GyrA inteins, using exteins that have similar insertionsites and therefore similar splicing active sites as in the nativecontext.

EXAMPLE II A M. Tuberculosis DnaB Intein-Mediated Positive SelectionSystem

[0088] A Positive Selection System using the Mtu DnaB Intein (IVPS) inits Native Mtu DnaB Extein in E. coli: Background

[0089] The hexameric E. coli helicase encoded by the dnaB gene interactswith an hexameric DnaC complex and ATP. Some DnaB mutants are dominantlethal (Bouvier and Oreglia, C. R. Acad. Sci. Hebd. Seances Acad. SciD., 280:649-652 (1975), Maurer and Wong, J. Bacteriol 170:3682-3688(1988), Saluja and Godson, J. Bacteriol. 177:1104-1111 (1995) andSclafani, et al., Mol. Gen. Genet., 182:112-118 (1981)). By dominant ordominantly cytotoxic, we mean that the toxicity occurs even ifhomologous proteins are present which are not cytotoxic or resistant tothe drug, i.e., the cytotoxic effect dominates irrespective of thepresence of non-cytotoxic homologs. The mutant protein is deficient inATP hydrolysis, helicase activity, and replication activity at thechromosomal origin of replication resulting in cell death (see FIG. 4A).Despite only moderate protein sequence identity between bacterialhelicases, arginine 231 is located in a conserved motif proposed tointeract directly with DnaC (Marszalek and Kaguni, J. Biol. Chem.,267:19334-19340 (1992) and Shrimankar, et al., J. Bacteriol.,174:7689-7696 (1992)). M. tuberculosis (Mtu) DnaB has a naturallyoccurring intein at the carboxy-terminus and an arginine at position 227homologous to arginine 231 of E. coli DnaB (see FIG. 4D).

[0090] We have demonstrated proficient protein splicing of the Mtu DnaBintein (IVPS) from the Mtu DnaB precursor protein in E. coli and alsohave shown that the R227C mutation results in dominant lethality.Therefore, a merodiploid cell containing a wild type dnaB gene and a MtuDnaB (R227C) gene is not viable unless protein splicing can be disrupted(see FIGS. 4B and 4C). By merodiploid we mean that the cell contains anextra copy of a gene (or several genes) which has been introduced intothe cell by any means known to one skilled in the art, such astransformation, infection, conjugation, plasmids, virus, phage, or bygenerating a transgenic strain and which may be present on either anepisomal element or on the host chromosome. The co-expression of achicken α-spectrin peptide library (as described in U.S. Pat. No.5,834,247 supra. at Example 17) allows for the positive selection ofpeptides that can disrupt splicing of the M. tuberculosis DnaB intein(see FIG. 5A). Likewise, this system can be used to screen for any agentthat inhibits splicing of the Mtu DnaB intein or any other DnaB inteinin vivo or for DnaB intein mutations that block splicing.

[0091] Construction of a Positive Selection System using the Mtu DnaBIntein (IVPS) in its Native Mtu DnaB Extein

[0092] As described in detail below, the Mtu dnaB gene has been clonedby PCR under T7 RNA polymerase transcriptional control. In E. coli, theMtu DnaB intein (IVPS) splices very efficiently from its naturalprecursor to produce the Mtu DnaB helicase. The Mtu dnaB gene has beenmutagenized at position 227 from arginine to cysteine and the plasmidtransformed into BL21 (DE3)-Gold (Stratagene, La Jolla, Calif.). In thepresence of the T7 RNA polymerase (induced by IPTG) only splicingdeficient clones can survive (see FIG. 4C).

[0093] First, the Mtu dnaB gene was cloned by PCR using M. tuberculosisH37Ra genomic DNA under the following experimental conditions. A forwardprimer 5′-AGGTGAGAA TTCATGGCGGTCGTTGATGACC-3′ (SEQ ID NO:37) and reverseprimer 5′-TATATAAAGCTTTCATGTCACCGAGCCATGTTGGCG-3′ (SEQ ID NO:38) wereused as described in the Extend Long Template PCR system (BoehringerMannheim, Indianapolis, Ind.) in the presence of 1×buffer 3 and 100 ngof M. tuberculosis genomic DNA. Amplification was carried out in aPerkin-Elmer/Cetus (Emeryville, Calif.) thermal cycler 480 for 2 min at94° C. and then cycled at 45° C., 30 sec; 68° C., 2 min; 95° C., 1 minfor 25 cycles. The PCR products of one 50 μl reaction and 5 μg of pET21a(Novagen, Madison, Wis.) were digested using 1000 U/ml of EcoRI and 800U/ml of HindIII in 1×EcoRI buffer (New England Biolabs, Inc., Beverly,Mass.). The digestion was performed at 37° C. for 1 hour. Digested PCRproducts and plasmid DNA were separated by agarose gel electrophoresisand the excised bands further purified using QIAEX II beads as describedby the manufacturer (Qiagen, Studio City, Calif.). Ligation was carriedout at 16° C. for 1 hour using a 1:5 ratio of vector to insert and40,000 U/ml of T4 ligase (New England Biolabs, Inc., Beverly, Mass.).Ligation products were transformed into E. coli strain Novablue DE3(Novagen, Madison, Wis.) competent cells. Recombinant plasmids werechecked by EcoRI/HindIII digestion which results in the excision of thecloned inserts. One of the resultant correct plasmids was named pEA807.The sequence of the dnaB insert was checked by DNA sequencing.

[0094] Second, the 1200 bp BgIII/SgrAI fragment from pEA682 containingthe spectrin-based peptide library was transferred to pEA807. PlasmidspEA682 and pEA807 were digested in 1×buffer 2 (New England Biolabs,Inc., Beverly, Mass.) using 500 U/ml of BgIII and 240 U/ml of SgrAl. Thedigestion was performed at 37° C. for 1 hour. Digested plasmids wereseparated by agarose gel electrophoresis and the excised bands furtherpurified using QIAEX II beads as described by the manufacturer (Qiagen,Studio City, Calif.). Ligation was carried out at 16° C. for 1 hourusing a 1:5 ratio of vector to insert and 40,000 U/ml of T4 ligase (NewEngland Biolabs, Inc., Beverly, Mass.). Ligation products weretransformed into E. coli strain ER2726 (New England Biolabs, Inc.,Beverly, Mass.) competent cells. Recombinant plasmids were checked byClal/Ncol digestion. One of the resultant correct plasmids was namedpEA808.

[0095] Third, the Mtu dnaB gene was mutagenized to R227C by PCR underthe following experimental conditions. A forward primer5′-AGGTGAGMTTCATGGCGGTCGTTGATGACC-3′ (SEQ ID NO:39) and reverse primer5′-TTTCCCACGCCCGGGCaCGCCGC CACGATGACC-3′ (SEQ ID NO:40) were used asdescribed in the Extend Long Template PCR system (Boehringer Mannheim,Indianapolis, Ind.) in the presence of 1×buffer 3 (New England Biolabs,Inc., Beverly, Mass.) and 500 ng of pEA808 DNA. Amplification wascarried out in a Perkin-Elmer/Cetus (Emeryville, Calif.) thermal cycler480 for 2 min at 94° C. and then cycled at 45° C., 30 sec; 72° C., 45sec; 95° C., 1 min for 20 cycles. The PCR products of one 50 μl reactionand 2 μg of pEA808 were digested overnight at 37° C. using 100 U/ml ofEcoRI and 40 U/ml of Srfl in the 1×PCR-Script reaction buffer(Stratagene, La Jolla, Calif.). Digested PCR products and plasmid DNAwere separated by agarose gel electrophoresis and the excised bandsfurther purified using QIAEX II beads as described by the manufacturer(Qiagen, Studio City, Calif.). Ligation was carried out at 16° C. for 1hour using a 1:1 ratio of vector to insert and 40,000 U/ml of T4 ligase(New England Biolabs, Inc., Beverly, Mass.). Ligation products weretransformed into E. coli strain XL10-Kan (Stratagene, La Jolla, Calif.)competent cells. Recombinant plasmids were checked by Ndel/Ascl. One ofthe resultant correct plasmids was named pEA809.

[0096] Fourth, the Notl dnaB-spectrin module was inverted on plasmidpEA809. 2 μg of pEA809 DNA was digested with 500 U/ml of Notl at 37° C.for 2 hours and digestion products split into two tubes. One tubecontaining 1 μg of Notl digested pEA809 was incubated further with 100U/ml Calf Intestinal Alkaline Phosphatase (CIP, New England Biolabs,Inc., Beverly, Mass.) for 20 minutes at 37° C. Digested plasmid DNA fromboth tubes was separated by agarose gel electrophoresis and the excisedbands further purified using QIAEX II beads as described by themanufacturer (Qiagen, Studio City, Calif.). Ligation of the vector bandfrom the CIP treated tube and the insert band from the CIP untreatedtube was carried out at 16° C. for 1 hour using a 1:1 ratio of vector toinsert and 40,000 U/ml of T4 ligase (New England Biolabs, Inc., Beverly,Mass.). Ligation products were transformed into E. coli strain XL1-Blue(Stratagene, La Jolla, Calif.) competent cells. Recombinant plasmidswere checked by Sacll digest. One of the resultant correct plasmids wasnamed pEA810.

[0097] Fifth, the laclq gene from pEA810 was removed and replaced by asmaller DNA fragment from pBR322. pEA810 and pBR322 DNA were digestedusing 500 U/ml EcoRV and 500 U/ml HindIII in buffer 2 (New EnglandBiolabs, Inc., Beverly, Mass.) at 37° C. for 2 hours. Digested plasmidDNA was separated by agarose gel electrophoresis and the excised bandsfurther purified using QIAEX II beads as described by the manufacturer(Qiagen, Studio City, Calif.). Ligation of the pEA810 vector band andthe pBR322 insert band was carried out at 16° C. for 1 hour using a 1:1ratio of vector to insert and 40,000 U/ml of T4 ligase (New EnglandBiolabs, Inc., Beverly, Mass.). Ligation products were transformed intoE. coli strain XL1-Blue (Stratagene, La Jolla, Calif.) competent cells.One of the resultant correct plasmids was named pEA813.

[0098] Sixth, the first Mtu R227C dnaB Aatll site of pEA813 waseliminated by site-directed silent mutagenesis using the QuickChange kit(Stratagene, La Jolla, Calif.) and oligonucleotides5′-GCCGCCGATCCGCGACATCGTAGATTTCGGCC-3′ (SEQ ID NO:41) and reverse primer5′-GGCCGAAATCTACGA TGTCGCGGATCGGCGGC-3′ (SEQ ID NO:42) resulting inplasmid pEA832.

[0099] Seventh, the wild type intein containing Mtu DnaB gene of plasmidpEA808 was shuffled back into pEA832. pEA808 and pEA813 DNA weredigested using 1×buffer 1 (New England Biolabs, Inc., Beverly, Mass.)with 500 U/ml EcoRI and 500 U/ml HindIII at 37° C. for 1 hour. Digestedplasmid DNAs were separated by agarose gel electrophoresis and theexcised bands further purified using QIAEX II beads as described by themanufacturer (Qiagen, Studio City, Calif.). Ligation of the pEA813vector band and the pEA808 insert band was carried out at 16° C. for 1hour using a 1:1 ratio of vector to insert and 40,000 U/ml of T4 ligase(New England Biolabs, Inc., Beverly, Mass.). Ligation products weretransformed into E. coli strain XL1-Blue (Stratagene, La Jolla, Calif.)competent cells. One of the resultant correct plasmids was named pEA825.

[0100] Eighth, the Aatll site elimination in pEA825 was performedidentically as described for pEA813, resulting in plasmid pEA835.

[0101] Screening for Peptides That Disrupt the Mtu DnaB intein (IVPS)Protein Splicing

[0102] The following is an actual experimental example demonstrating theuse of this system to select for peptides that block splicing. Asdetailed below, random peptides of 7-12 amino-acids were insertedin-frame into the loop of the 2 EF-hand motif of α-spectrin, contained,as described above, on the same plasmid as the Mtu DnaB intein (IVPS)selection system. The resulting plasmids were electro-transformed intothe T7 RNA polymerase E. coli strain ER2744 (New England Biolabs, Inc.,Beverly, Mass.) (see FIGS. 5A and 5B). Transformants were selected in LBliquid growth media in the presence of ampicillin and IPTG to allowselection against the splicing proficient clones. Plasmid DNA wasisolated from selected clones and digested to isolate DNA fragmentsencoding the selected spectrin peptides. The selected spectrin loopregion DNA was cloned back into the original selection plasmid.Iterative rounds of selection and “back-cloning” were performed (FIG.5B). After selection, the selected spectrin peptide were cloned intopEA825 (containing the non-toxic DnaB gene) for expression analysis.Final selected clones were grown individually in liquid culture and theplasmid-encoded Mtu dnaB gene specifically induced by IPTG. Crudeprotein cell extracts were electrophoresed and blotted forimmuno-staining. Clones in which the Mtu DnaB spliced product was notdetected were considered positives, i.e. clones in which splicing hadbeen disrupted, potentially by a selected peptide.

[0103] The random peptide library was synthesized in vitro using thefollowing protocol. A single strand/double strand DNA hybrid cassettewas synthesized by annealing of 2 oligonucleotides:5′-TGTCAAGAATCGATTCAMTTCGGGGTC AGGCTCTCC((W)NN)₇₋₁₂ATAGCCMGCGATCGCAGGCAGCTTTT AAAGCCCTGATGGTTCAGACGT-3′ (SEQ ID NO:43) and5′P-CTGAACCATCAGGGC-3′ (SEQ ID NO:44). 5 μg of oligonucleotide SEQ IDNO:43 and 3 molar equivalents of oligonucleotide SEQ ID NO:44 were mixedtogether in the presence of 0.1 M NaCl in a final volume of 50 μl. Themixture was boiled and immediately cooled down to room temperature inthe same boiler. The single strand random nucleotide part of the DNAhybrid cassette formed by annealing of the 2 oligos was extended using400 μM of each dNTPs and 60 U/ml of Klenow DNA polymerase (New EnglandBiolabs, Inc., Beverly, Mass.) in a final volume of 200 μl in 1×EcoPolbuffer (New England Biolabs, Inc., Beverly, Mass.). The extensionreaction was incubated 20 minutes at 37° C. and further purified usingQIAEX II beads as described by the manufacturer (Qiagen, Study City,Calif.). 60 μg of pEA832 (50 μg/ml) were digested in 1×Buffer 4 (NewEngland Biolabs, Inc., Beverly, Mass.) using 400 U/ml of Aatll (NewEngland Biolabs, Inc., Beverly, Mass.) and 500 U/ml of Clal (New EnglandBiolabs, Inc., Beverly, Mass.) in the presence of 100 μg/ml of BSA. Thedigestion was performed at 37° C. for 2 hours. Synthesized randomcassettes (20 μg/ml) were digested in 1×Buffer 4 (New England Biolabs,Inc., Beverly, Mass.) using 500 U/ml of Clal (New England Biolabs, Inc.,Beverly, Mass.) in the presence of 100 μg/ml of BSA. Cassettes werefurther purified using QIAEX II beads as described by the manufacturer(Qiagen, Studio City, Calif.). Digested plasmid DNA was electrophoresedon a 0.7% agarose gel and the excised bands further purified using QIAEXII beads as described by the manufacturer (Qiagen, Studio City, Calif.).Ligation was carried out at 16° C. for 1 hour using a 1:1 ratio ofvector (2 ng/μl) to insert and 1,600 U/ml of T4 ligase (New EnglandBiolabs, Inc., Beverly, Mass.). Ligation products wereelectro-transformed into E. coli strain ER2744 (New England Biolabs,Inc., Beverly, Mass.) competent cells (competency of 1×10⁹ pEA835transformants/μg) using 2 μg of total ligation product for each 200 μlaliquot of competent cells, at 2.5 kV/cm in a 2 mm cuvette (BIORAD,Richmond, Calif.). Cells were allowed to recover in a shaker for 1 hourat 37° C. Recovered transformants were inoculated at 1/100 dilution intoLB liquid growth media containing 100 μg/ml of ampicillin and 1 mM IPTG.Transformants were incubated overnight at 30° C. Plasmid DNA wasisolated from the overnight culture using tip100 columns (QIAGEN, StudioCity, Calif.)), Aatll/Clal digested as above and electrophoresed on a 4%GTG Nusieve agarose gel (FMC BioProducts, Rockland, Me.). The 57 to 72bp spectrin loop DNA inserts were purified using QIAEX II beads asdescribed by the manufacturer (Qiagen, Studio City, Calif.) and clonedback into Aatll/Clal digested and purified selection plasmid (pEA832) asdescribed above. This protocol was repeated 3 times to enrich the poolof transformants for peptide clones having the most biologically activesequences against the protein splicing of the Mtu DnaB intein (IVPS).Finally selected spectrin modules were cloned into a pEA832 homologousplasmid containing the wild type Mtu dnaB gene (pEA825) and grownindividually in 10 ml LB containing 100 μg/ml ampicillin at 37° C. andinduced with 1 mM IPTG for 3 hours. Crude protein cell extracts wereelectrophoresed on a 10-20% gradient gel (Novex, San Diego, Calif.). Thegel was further electro-blotted for immuno-staining using anti-T7 tagantibodies (Novagen, Madison, Wis.) to detect Mtu DnaB protein splicingproducts (FIG. 5C). Lane Eco DnaB contains extracts of T7-tagged E. coliDnaB without the intein. pMtuDnaB contains extracts from a cloneexpressing only Mtu DnaB. Lanes p814, p815, p816, p817, and p818 containextracts of the clones pEA814, pEA815, pEA816, pEA817, and pEA818,respectively, encoding peptides selected for inhibition of splicing. Todemonstrate that the inhibition of splicing was due to the peptideinserted into the chicken α-spectrin loop, the selected sequence ofpEA818 was replaced with the spectrin sequence, DLPMVEE (SEQ ID NO:10)to generate clone pEA818rev, and extracts loaded on lane p818rev.Splicing of pEA818rev occurred as efficiently as with the pMtu DnaBclone that expresses the wild-type spectrin protein. Note that in theabsence of splicing, much of the DnaB precursor undergoes cleavage atthe intein C-terminal splice junction.

[0104] The sequence of the inserted peptides in these clones is asfollows: pEA814 TVQSTKR (SEQ ID NO:5) pEA815 RPAPRPL (SEQ ID NO:6)pEA816 PTARTYE (SEQ ID NO:7) pEA817 PTRPTAPPLNFS (SEQ ID NO:8) pEA818HPNPHPTLSGQR (SEQ ID NO:9) pEA818rev DLPMVEE (SEQ ID NO:10)

[0105] We have thus demonstrated that this system can be used to selectfor peptides that block splicing of the Mtu DnaB intein. This system isamenable to selection of any modulators of splicing of the Mtu DnaBintein or other DnaB inteins, as long as the agent can enter a cell.

EXAMPLE III In Vivo Control of Protein Splicing for ChemotherapeuticPurposes or to Make Controllable Gene Knockouts

[0106] The selection and screening systems described for selection ofagents that modulate protein splicing can also be applied to intein-lessversions of the extein gene to select for agents that inhibit oractivate the extein gene product. All of the selection and screeningsystems described in this patent are based on the activity or inactivityof the extein portion of the precursor. If one deletes the intein fromthe intein-containing gene by methods known to one skilled in the art,then one can select for agents that block or activate extein activityalso using the methods described for inhibiting or activating splicingof the intein containing precursor, since these latter methods involveassaying extein function. For example, if one deletes the intein fromthe Mtu DnaB gene by methods known to one skilled in the art, then onecan select for agents that block activity of the cytotoxic Mtu DnaBprotein using the methods described for inhibiting splicing of the DnaBintein. M. tuberculosis can then be attacked using a cocktail of twoagents that block activity of the essential DnaB protein, making it moredifficult for the organism to develop resistance to these agents.

[0107] We have previously described the insertion of a CIVPS or IVPSinto a foreign gene. In these cases, protein splicing could becontrolled by temperature, mutation, pH, photo-activated blockinggroups, phosphorylation or peptides (Comb, et al., U.S. Pat. No.5,834,247 and Comb, et al., U.S. Pat. No. 5,496,714). In this Example wedescribe a general method for selecting specific protein splicinginhibiting or activating agents that are capable of controlling proteinsplicing in vivo or in vitro. The methods are equally applicable togenetic selection systems or reporter systems. By genetic selection, wemean, in this Example, that viability or growth rate of the testorganism is monitored during the experiment, while a reporter system inthis Example refers to the monitoring of a marker, such as colordetection, fluorescence, phenotype, etc., rather than cell viability.Genetic selection or reporter systems are used to identify agents thatcan either disrupt or catalyze protein splicing of a given intein,depending on the context of the experiment. Any genetic selection orreporter system known to one skilled in the art can be used to isolateagents which disrupt or catalyze protein splicing. This strategy isequally applicable to any intein present in a foreign context or in itsnative or homologous context (e.g., the insertion of an intein at thesame position in an homologous extein). However, use of the nativeextein is preferable because it best represents the enzyme target of theintein. If the native precursor does not express well or splice well inthe experimental host organism, then the intein can be inserted into thesame site in that host organism's homolog of the native extein or inanother extein homolog with desired properties for testing, using anymethod known to one skilled in the art or described in the previousExamples. This method of finding agents that modulate splicing isapplicable to any host, as long as the protein splicing precursor isoperably linked to the appropriate control signals for transcription andtranslation in that host. As the target organism may not be an easyexperimental model for identifying agents that modulate proteinsplicing, the agent may first be identified in a model system and thentested in the final target organism. This strategy is summarized in FIG.8.

[0108] Experiments involving inhibition of splicing start with aprecursor that contains a fully active intein that may or may not becontrollable. The goal of this experiment is to find agents that can beused to control splicing of this intein. In experiments involvingactivation of splicing, a CIVPS (controllable intein) or an inactiveintein is required, as the goal is to find agents that activate thepreviously inactive intein. The intein may be inactivated by any meansknown to one skilled in the art, such as temperature sensitive inteins,inteins with mutations in amino acids known to be involved in catalysisthat slow down or block splicing (including the conserved amino acids atboth splice junctions and in intein Block B, (Perler, Nucleic Acids Res.25:087-1093 (1997), Pietrokovski, Protein Sci., 3:2340-2350 (1994))inteins which have been randomly mutated and selected for inhibition orblockage of splicing.

[0109] A positive selection system is preferred. In general, a positiveselection system consists of a gene that is detrimental to a hostorganism depending on the growth media or the host strain geneticbackground. The gene product is static or lethal for the cell, killingthe host or preventing growth unless the gene product is inactivated.The gene product may be directly cytotoxic to the host in a dominantmanner, as in the DnaB example (Example II) or it may be dominantlycytotoxic in response to a drug which the chromosomal copy of the geneis resistant to, as in the GyrA example (Example I). By dominant ordominantly cytotoxic, we mean that the toxicity occurs even ifhomologous proteins are present which are not cytotoxic or resistant tothe drug, i.e., the cytotoxic effect dominates irrespective of thepresence of non-cytotoxic homologs. In the context of a protein splicinginhibition system, positive selection involves a system that allowsselection against the splicing of an IVPS or intein. If splicing occurs,the cytotoxic extein protein will be active and kill the cell or inhibitgrowth; if splicing is disrupted, the cytotoxic extein protein will beinactive and cells will grow. Cell growth can be monitored by any meansknown to one skilled in the art, including, but not limited toobervation of a colony on solid media, optical density, monitoring offluorescent reporters of cell growth such as green fluorescent proteinof luciferase activity. The extein gene may be an unrelated reportersystem or the natural extein of the intein (either using the naturalprecursor or inserting the intein into a homologous extein context)(FIG. 8). In this context, selection systems have the advantage thatonly agents that inhibit splicing allow cell growth and are thus easilyfound amongst the background of agents that have no effect on splicingor are directly toxic to the cell. If the agents to be tested are alsoexpressed in the host cell, then one examines the colonies that surviveon the plate. If the agent to be tested is not expressed in the hostcell, but is instead added to the media, then aliquots of host cellsmust be arrayed for testing with individual agents or pools of agents inany number of devices, such as microtiter dishes. In such cases, cellgrowth may be more easily measured if the cells expres a protein thatleads to fluorescence, such as green fluorescent protein or luciferase.

[0110] When selecting for agents that activate splicing, the intein isalready present or is inserted into a gene whose protein product isrequired for cell growth. In the absence of splicing, the cell fails togrow or dies. In order to practically employ this selection system, asecond gene is present which can rescue the cell in the absence ofsplicing. This second copy of the gene should be controllable, bymethods such as a temperature sensitivity or controllable promoters, toallow cell growth until the agent which activates splicing is applied orinduced in the cell. The cells are treated with the splicing activatorand then moved to the nonpermissive condition for activity of the secondgene product that does not contain the intein or expression of this geneis turned off. Cell growth will then require splicing since the secondgene product lacking the intein is no longer active.

[0111] Another method for identifying agents that modify proteinsplicing involves screening rather than genetic selection. Screeningsystems employ reporter genes whose products can be readily assayed, butdo not necessarily affect cell growth. Many reporter systems are known,such as the blue/white β-galactosidase screening system. β-galactosidaseacts on X-gal, for example, to generate a blue color; in the absence ofβ-galactosidase activity, the X-gal remains uncolored or ‘white’. Otherreporters include those described in Burns and Beacham, Gene, 27:323-325(1984) and Mechulam, et al., J. Bacteriol., 163:787-791 (1985). One canuse native precursors if reporter systems are available for those exteingenes or the intein can be cloned into the reporter gene(β-galactosidase in this Example) (see for example, Belfort, U.S. Pat.No. 5,795,731 and Comb, et al., U.S. Pat. No. 5,834,247). Agents thatinhibit splicing of an otherwise active intein will block reporterprotein functions, such as β-galactosidase action on X-gal, resulting inwhite instead of blue clones. Agents that activate splicing of anotherwise inactive or slowly acting intein restore reporter proteinfunctions, resulting in blue clones using the β-galactosidase system asan example. If the agents to be tested are also expressed in the hostcell, then one examines the colonies that survive on the plate. If theagent to be tested is not expressed in the host cell, but is insteadadded to the media, then aliquots of host cells must be arrayed fortesting with individual agents or pools of agents in any number ofdevices, such as microtiter dishes. Unlike selection, all cells grow inreporter systems and one must determine whether the read out is positiveor negative for each colony or microculture.

[0112] Previous Examples have described genetic selection systems basedon the pheS non-homologous selection system and the gyrA and dnaBintein/extein systems. This Example describes how one would screen foragents that modulate splicing using any selection or reporter system.Note that the selection or screening systems may not have beenoriginally identified in the organism containing the intein. However, ifa selection or screening system has been described for the exteinhomolog, it can be adapted to the intein containing homolog. As in thecase of DnaB, the same mutation can be made in the intein containinghomolog to generate a selectable phenotype for the intein containingextein gene. As in the case of GyrA, the screening system can involve achromosomal mutation that leaves the host resistant to a drug; all thatneed be done is to show that the intein containing homolog is alsosensitive to the drug.

[0113] Iterative screening (FIG. 5B) provides a method of identifyinglead compounds and reducing background and can be used in any of theschemes described below. Iterative screening involves repeated cycles oftesting of the agent on fresh extein genes. It helps insure that theagent is not acting on a mutated extein, which could also be aby-product of screening.

[0114] Positive Selection Systems for Inhibition or Activation ofProtein Splicing of an Intein in its Natural Precursor or an ExteinHomolog

[0115] In this case, the intein of interest is naturally found in atarget gene which can naturally serve as a selectable marker or reporteror which can be converted into a selectable marker or reporter. Initialexperiments may be performed in the target organism or an experimentallymore amenable model host such as bacteria, E. coli, yeast, mammaliancells, insect cells, etc. The decision as to whether to use the naturalsplicing precursor to select for agents that block splicing or to firstinsert the intein gene into a homologous extein gene from a modelorganism depends on the similarity amongst the extein genes, the abilityof the natural precursor or recombinant precursors to express in themodel hosts used for selection or screening, and the ability of eachprecursor to splice in the model hosts. (FIG. 8) These parameters willhave to be experimentally determined, although the more similar theextein sequences, the more likely that splicing will work in thehomologous extein protein from the model organism. Sequence comparisonwill indicate the appropriate homologous intein insertion site in thehomologous extein gene from the model organism.

[0116] Next, one has to determine by a literature search whether anygenetic selection systems or screens are available for the target exteinin any organism and whether the extein gene is essential for cell growthin any organism. If the target gene is essential, but no geneticselection or screens are available, it can be mutagenize directly or inmodel systems to attempt to generate a selection or reporter system. Ifthe target gene product is essential to the cell, under definedconditions, the host gene can be either knocked out and replaced by acontrollable copy of the gene or mutated to generate a temperaturesensitive activity. The intein containing gene must the produce anactive product when the host gene homolog is inactivated. A temperaturesensitive phenotype can easily be generated by random or rationalmutation by one skilled in the art. Once a selection system has beenidentified and the best splicing precursor has been determined(selecting from the naturally occurring precursor, or after insertingthe intein into the homologous extein from the target or selectionorganism), testing for agents that block splicing can begin in either amodel organism or the target organism, depending on ease of use. Some ofthe possible schemes for identifying agents that block or activatesplicing are shown in FIG. 9.

[0117] Scheme 1 is a method for selecting for agents that inhibitsplicing. The selection system involves a dominant cytotoxic phenotypein response to a drug. By dominant cytotoxic we mean that the splicedproduct is toxic to the cell irrespective of expression of a resistantcopy of the extein gene. The GyrA system described in Example I is anexample of this type of scheme. The selection host organism contains achromosomal copy of the extein gene that is resistant to the drug andallows growth of the organism in the presence of the drug. First, amerodiploid is made containing a gene which is sensitive to the drug andcontains the intein, and a gene which is resistant to the drug and doesnot contain an intein. Second, the host containing the resistant exteingene and the intein containing sensitive extein gene is then treatedwith agents that can enter the cell or by induction of expression ofagents within the cell. Finally, the selection drug is added to thecells. If the intein splices, the drug sensitive target protein killsthe cell or inhibits growth when the drug is present. If any agentblocks splicing, no drug sensitive extein protein is made and theorganism grows. Usually, one tests a library-of compounds of any type,rather than a single agent, and one uses small cultures, as inmicrotiter dishes, for example. Any type of agent can be used, as longas it can enter the cell. Alternatively, the agent can be cloned andexpressed in the target cell and clones can be tested for growth onplates or in liquid media. Expression of combinatorial peptide librarieswould be an example of such an agent that is expressed in the cell.

[0118] Scheme 2 is a second method for selecting for agents that inhibitsplicing. The selection system involves a dominant lethal phenotype inthe absence of exogenous drug treatment that is inherent in the inteincontaining extein protein or can be introduced into the extein protein.The DnaB system described in Example II exemplifies this type of system.The selection host organism contains a wild type gene that is not toxicto the cell and allows growth of the organism. First, a mero-diplid ismade containing a gene which is toxic to the cell, but contains anintein and an intein-less extein gene which is not toxic. Next, thishost is treated with agents that can enter the cell before the cytotoxicprecursor gene is expressed. Finally, expression of the inteincontaining cytotoxic extein gene is induced. If the intein splices, thecytotoxic target protein kills the cell or inhibits growth. If any agentblocks splicing, no cytotoxic target protein is made and the organismgrows. Usually, one tests a library of compounds of any type, ratherthan a single agent, and one uses small cultures, as in microtiterdishes, for example. Any type of agent can be used, as long as it canenter the cell. Alternatively, the agent can be cloned and expressed inthe target cell and clones can be tested for growth on plates or inliquid media. Expression of combinatorial peptide libraries would be anexample of such an agent that is expressed in the cell.

[0119] Scheme 3 selects agents that inhibit splicing of an essentialgene. In this case, the chromosomal copy of the gene, or its equivalent,is either temperature sensitive, sensitive to a drug in a recessivemanor, or under some type of expression control. Alternatively, thechromosomal copy of the extein gene is inactivated or knocked out. Thecells can grow under conditions where the gene product is not needed.The cells are then shifted to conditions which require the exteinprotein for survival. An example of this type of extein is a metabolicenzyme. When cells are grown in rich media, they can grow. However, whencells are grown in minimal media or media lacking the downstream productof the extein blocked metabolic pathway, the cells fail to grown. Theintein containing target gene is not temperature sensitive or isresistant to the drug. If splicing occurs under non-permissiveconditions for the chromosomal extein homolog, then the cells live. Thissystem requires assay of cell growth in isolated containers, such asmicrotiter dish wells, for example. If the agent blocks splicing, thenthe cells will not grow under non-permissive conditions for activity ofthe intein-less copy of the extein protein. Cell growth can bedetermined by any means known to one skilled in the art, including, butnot limited to measuring optical density or presence of a fluorophoregenerated in the cell. First an experimental host must be found thatcontains a controllable copy of the extein gene or its equivalent. It ispropagated under permissive conditions for expression of activeintein-less extein protein. Second, this host is transformed with avector containing a wild type extein gene or extein homolog genecontaining the intein. Third, merodiploid cells containing theintein-plus and intein-minus copies of the extein gene, or itsequivalent, are treated with agents to block splicing and are alsoshifted to non-permissive conditions for activity of the intein-lessextein protein. This may involve a shift to a temperature at which theintein-minus protein is inactive, removal of inducers for expression ofthe intein-minus shifting to different media, or addition of a drugwhich inactivates the intein-minus protein. If splicing occurs, thecells will continue to grow using the intein-plus gene product. However,if the agent inhibits splicing, products of both copies of the gene areinactivated and the cells die. Alternatively, the agent can be clonedand expressed in the target cell. However, in this case, each clone mustbe copied or replica plated to maintain a living copy of the library anda copy to be tested for inhibition of splicing. Expression ofcombinatorial peptide libraries would be an example of such an agentthat is expressed in the cell.

[0120] Schemes 4-6 are methods of selecting for agents that activatesplicing rather than inhibit it. The precursor contains an inactiveintein which is introduced into the cell on any type of vector. Theagent(s) may be added individually or in pools to isolated cultures.Alternatively, the agent can be cloned and expressed in the target cell.However, in this latter case, each clone must be copied or replicaplated to maintain a living copy of the library and a copy to be testedfor activation of splicing. Expression of combinatorial peptidelibraries would be an example of such an agent that is expressed in thecell.

[0121] Scheme 4 is identical to scheme 1. In the presence of the drug,an agent that activates splicing kills the host since the intein-plusdrug sensitive copy of the gene is active and dominantly cytotoxic. Oneassays for the absence of growth in isolated cultures, such asmicrotiter dish wells, for example.

[0122] Scheme 5 is similar to scheme 1. An agent that activates splicingkills the host since the dominantly cytotoxic extein is active aftersplicing of the intein, irrespective of the presence of the wild typeextein protein derived from the intein-minus gene. One assays for theabsence of growth in isolated cultures, such as microtiter dish wells,for example.

[0123] Scheme 6 is similar to scheme 3, except that the selection systemrequires expression of the spliced target gene for cell growth andselects for agents that activate splicing. In this type of system, theintein-minus copy of the target extein gene, or its equivalent, iseither temperature sensitive, sensitive to a drug in a recessive manor,or under some type of expression control. Alternatively, the chromosomalcopy of the extein gene is inactivated or knocked out. The cells cangrow under conditions where the gene product is not needed. The cellsare then shifted to conditions which require the extein protein forsurvival. An example of this type of extein is a metabolic enzyme. Whencells are grown in rich media, they can grown. However, when cells aregrown in minimal media or media lacking the downstream product of theextein blocked metabolic pathway, the cells fail to grow. The inteincontaining target gene is not temperature sensitive or is resistant tothe drug. The target gene containing the intein is introduced into thecell by any means known to one skilled in the art. In this case, theintein has been modified so that it can not splice under the assayconditions. The host copy of the gene is expressed in an active formunder permissive conditions (permissive temperature, in the absence ofdrug, rich media under permissive expression conditions, etc.), allowingthe cells to grow. The intein-plus copy of the target extein gene,containing the inactive intein, is introduced into the cell. Afterexpression of the intein precursor is established, agents are addedexternally or peptide libraries are expressed internally to inducesplicing. After allowing the agent to activate splicing, the cells areshifted to the nonpermissive condition (non-permissive temperature, inthe presence of drug, minimal media under non-permissive expressionconditions, etc.). The only cells that can grow are those in whichsplicing activity has been restored by the agent. If an external agentis to be tested, then the agent is added to cells in isolatedcontainers, such as microtiter dish wells. Alternatively, the agent canbe cloned and expressed in the target cell. In this case, the library ofagents can be directly tested for cell viability on plates. Expressionof combinatorial peptide libraries would be an example of such an agentthat is expressed in the cell.

[0124] Reporter Systems for Inhibition or Activation of Protein Splicingof an Intein in its Natural Precursor or an Extein Homolog

[0125] Any extein that can be converted into a tractable phenotype canbe used in a reporter system screen. This type of system requires theability to differentiate between active and inactive extein by anydirect or indirect means. Once the reporter system is available, theintein containing gene is introduced into the cell by any method knownto one skilled in the art and agents that inhibit splicing are added orinduced as above. Alternatively, an inactive intein is introduced into acell and agents that activate it are added or induced as above. One thenexamines individual clones and determines whether the extein is activeor not.

[0126] Systems for Inhibition or Activation of Protein Splicing of anIntein in an Unrelated Extein Context

[0127] The scenario for this method of identifying agents that inhibitor activate splicing is the same as schemes described above, except thatthe intein is placed in an unrelated extein. However, one must firstdetermine that the intein splices in the non-homologous extein (FIG. 8).To improve the probability that an intein will splice in anon-homologous foreign context, the intein insertion site should be assimilar to the natural extein sequence as possible for at least 1 and upto 5 or more extein residues. If the intein is inserted into anonessential region of the target protein, one could possibly modify thesequence of the target protein at the intein insertion site to be thesame as the native extein sequence of that intein. The intein must becloned prior to a Ser, Thr or Cys with the amino acid naturallyfollowing the intein being the best choice or the Ser, Thr or Cys codonmust be inserted into the extein along with the intein sequence. Toimprove folding, surface locations on the protein would be preferablesince they are more likely to allow the extein to fold independently ofthe intein. If the structure of the target protein is unknown, proteasesensitive sites on the target protein should be good positions to insertthe intein.

[0128] Since splicing can be sequence dependent, it is optimal toexperimentally identify agents that modify splicing in the same targetprotein that one wants to finally control. However, agents couldpossibly also control splicing of that intein in any extein. New exteinsmay have to be treated experimentally.

[0129] Controllable Knockouts

[0130] Once an agent has been found which can inhibit or activatesplicing, the homologous extein gene in the target organism is replacedby the homologous gene containing the intein by methods known to oneskilled in the art. For example, this may be performed in a one stepprocess by inserting the intein-containing gene directly into thechromosomal copy of the extein gene by homologous recombination.Alternatively, the intein containing gene is introduced into theorganism and the non-intein containing homolog is inactivated eitherconcurrently or separately and in any order of event. Once the only copyof the active extein gene contains a intein, gene function can beinhibited if the organism is treated with an agent that blocks splicing.On the other hand, if a splicing impaired intein is used, gene functioncan be activated if the organism is treated with an agent that activatessplicing. The agents and splicing can be modulated at any time duringthe development and life of the organism by addition or removal of thesplicing activating or inhibiting agent. For example, a gene for mouseembryogenesis can be replaced by an intein containing gene homolog andthe product of that gene can be activated or inactivated at varioustimes to determine when the gene product is required and if it isrequired during multiple stages of development or growth. In a secondexample, a gene product thought to be required for passage through aspecific stage of the cell cycle could be replaced with an inteincontaining copy that would allow study of exactly when the gene productis required or to synchronize the culture by arresting all cells at thesame point in the cell cycle to study the effect of any agent, etc., ona synchronized culture of cells.

[0131] Use Of Controllable Inteins (CIVPS) in Therapeutics

[0132] Several options can be envisioned for the use of controllablesplicing to deliver active proteins at specific times or to specificplaces. In many instances, therapeutic drugs can be cytotoxic to thehost and would be best if only active at the target site. For example,chemotherapy drugs are often generally cytotoxic and adverse reactionsin normal cells could be eliminated if the drug could be specificallyactivated in the tumor. If one has a drug that is at least partiallyproteinacious, an intein that can be activated or inhibited by a secondagent, as described above, could be inserted into the protein portion ofthe therapeutic agent. The drug is then administered systemically in aninactive form. The drug could then be specifically activated in thetumor or target organ by (1) injecting the activating agent into thetumor, (2) exposing the tumor to laser treatment to increase thetemperature of the tumor and thus induce splicing of a temperaturesensitive intein, (3) use gene therapy to target the inactive cytotoxicprecursor to the tumor cells and then add the splicing activatorsystemically or (4) use gene therapy to target the activating peptide tothe tumor and add the inactive intein containing drug systemically. Inthe Examples described above, the inactive cytotoxic precursor or theactivating peptide, respectively, could be transformed systemically witha vector that is not tissue or cell specific, and only expressed inspecific target cells by operably linking these genes to tissue specificpromoters.

EXAMPLE IV Methods for Generating Temperature Controllable Inteins

[0133] The methods used for identifying agents that inhibit or activatesplicing can also be used to identify inteins that are active at onetemperature and inactive at a second temperature (referred to astemperature sensitive inteins). Instead of adding an external agent orexpressing an internal agent, the intein is randomly mutated by anymethod known to one skilled in the art, such as error prone polymerasechain reaction (FIG. 10) or use of combinatorial DNA sequences atspecific regions in the intein. Alternatively, one can specificallymutate residues thought to function in or assist the chemical reactions,such as the C-terminal splice junction residues, the intein N-terminus,the intein penultimate residue, the residues in intein Block B (Perler,Nucleic Acids Res., 25:1087-1093 (1997); Perler, Nucleaic Acids Res.,27:346-247 (1999); Pietrokovski, supra), residues proximal to the inteinactive site as determined crytallographically (Duan, et al., Cell,89:555-564 (1997); Klabunde, et al., Nat. Struct. Biol., 5:31-36(1998)), etc. The mutated intein gene is then introduced into a cell andexamined for the ability to splice under permissive and non-permissivetemperatures as chosen by the researcher, and can be any combination oftemperatures (FIG. 11). Splicing is assayed as in Examples I through IIIas long as the chromosomal or intein minus extein gene is not similarlytemperature sensitive.

[0134] Using the Mxe GyrA intein in the E.coli GyrA extein andexpressing the fusion in E.coli cells (Example I), we have identifiedseveral polymerase chain reaction generated mutations that rendersplicing of the Mxe GyrA intein temperature sensitive (FIGS. 10, 11, 12and 13). These precursors splice at 19° C., but not at 37° C. Moreover,these mutations concentrate in the beta-sheet that includes intein BlockB (FIGS. 12 and 13).

[0135] Screening for Temperature Sensitive Mxe GyrA Intein Mutants

[0136] The gyrA selection system described in Example I, can also beused to screen for temperature sensitive splicing mutants of the MxeGyrA intein in the ofloxacin sensitive E. coli GyrA extein. Experimentswere performed with a vector similar to pEA600. A splicing proficientclone and a splicing deficient clone (containing mutation of the inteinCys1 to Ala and Asn198 to Ala) were plated on solid media containingvarious concentrations of ofloxacin to determine the appropriate drugconcentration to allow growth of the splicing deficient clone whileblocking growth of the splicing proficient clone. The Mxe gyrA inteingene was then amplified by PCR (FIG. 10) using mutagenic strategiesknown to one skilled in the art and inserted into the E. coli gyrA gene.Libraries were plated on solid media containing ofloxacin at thepredetermined concentration, replica plated and grown at either 37° C.or 16° C. (FIG. 11). Only splicing defective clones survived and grew onthe plates. The replica plates were compared to identify clones thatgrew at 37° C., but not at 16° C. Such clones were picked and retestedfor temperature dependent splicing. Alternatively, the libraries ofmutated Mxe GyrA inteins in E. coli GyrA were grown at 37° C. and thenstreaked onto a second plate to test for lack of growth at the splicingpermissive temperature of 16° C. Splicing of the GyrA precursor wasexamined in clones that failed to grow at 16° C. by incubating in theabsence of ofloxacin at 37° C. for 3 hours and then shifting to 16° C.overnight. Cell lysates were electrophoresed in SDS-PAGE gels that werethen stained with Coomassie blue. Spliced GyrA was observed in severalclones, although splicing was not complete (FIG. 12).

[0137] The Mxe gyrA intein gene was sequenced from several of thesetemperature sensitive clones and found to have one or more mutationswhich are summarized in FIG. 13. The 3-D structure of the Mxe GyrAintein is known (Klabunde, et al., Nature Struct. Biol. 5:31-36(1998))., GyrA enabling us to place these mutations on the Mxe GyrAintein structure (FIG. 14). We found that many of the mutations were inthe beta-sheet including intein Block B (FIGS. 13 and 14), specificallyin Mxe GyrA intein beta-strand B8 and the loop between beta-strands B8and B9 (Klabunde, supra; Perler Cell 92:1-4 (1998)). Intein Block Bcontains conserved intein residues thought to assist in theautocatalytic reactions at the intein N-terminal splice junction(Klabunde, supra; Noren, C. J., et al. Angewandte Chemie (in press)).Mutation in residues proximal in space to intein Block B, as found inthis selection for temperature sensitive Mxe GyrA intein mutants, mayslightly perturb the position of Block B residues, resulting in thetemperature sensitive phenotype.

[0138] We suggest that mutation of the amino acids in the analogousbeta-strand and loop in other inteins may generate temperature sensitivemutants of any intein. Homologous regions in other inteins can be easilyidentified due to the structural similarity of known intein splicingdomains and intein multiple sequence alignments. To date, the 3-Dstructure of the Mxe GyrA intein (Klabunde, supra), the Sce VMA intein(Duan, et al., Cell 89:555-564 (1997)) and the Drosophila hedgehogprotein autoprocessing domain (Hall, et al. Cell, 91:85-97 (1997)) havebeen determined. The splicing domain of both inteins and the N-terminalpart of the hedgehog autoprocessing domain have the same protein fold;the alpha carbon trace of most of the amino acids in each of these 3structures are superimpossible (Klabunde, supra; Perler, supra (1998)).Intein amino acid sequence similarity comparisons have also beendescribed in the literature (Perler supra (1997), Pietrokovski,supra(1994), Pietrokovski, Protein Sci. 7:64-71 (1998), Dalgaard, etal., J. Comp. Biol. 4:193-214 (1997)).

[0139] Given the similarity in intein splicing domain structure andsequence, one skilled in the art should easily be able to identifyregions in any intein that are analogous to the Mxe GyrA inteinbeta-strand B8 and the loop between beta-strands B8 and B9, and usingthis information, mutate this region to specifically generatetemperature sensitive protein splicing mutants.

1 46 1 186 PRT Escherichia coli Gyrase A 1 Met Ser Asp Leu Ala Arg GluIle Thr Pro Val Asn Ile Glu Glu Glu 1 5 10 15 Leu Lys Ser Ser Tyr LeuAsp Tyr Ala Met Ser Val Ile Val Gly Arg 20 25 30 Ala Leu Pro Asp Val ArgAsp Gly Leu Lys Pro Val His Arg Arg Val 35 40 45 Leu Tyr Ala Met Asn ValLeu Gly Asn Asp Trp Asn Lys Ala Tyr Lys 50 55 60 Lys Ser Ala Arg Val ValGly Asp Val Ile Gly Lys Tyr His Pro His 65 70 75 80 Gly Asp Ser Ala ValTyr Asp Thr Ile Val Arg Met Ala Gln Pro Phe 85 90 95 Ser Leu Arg Tyr MetLeu Val Asp Gly Gln Gly Asn Phe Gly Ser Ile 100 105 110 Asp Gly Asp SerAla Ala Ala Met Arg Tyr Thr Glu Ile Arg Leu Ala 115 120 125 Lys Ile AlaHis Glu Leu Met Ala Asp Leu Glu Lys Glu Thr Val Asp 130 135 140 Phe ValAsp Asn Tyr Asp Gly Thr Glu Lys Ile Pro Asp Val Met Pro 145 150 155 160Thr Lys Ile Pro Asn Leu Leu Val Asn Gly Ser Ser Gly Ile Ala Val 165 170175 Gly Met Ala Thr Asn Ile Pro Pro His Asn 180 185 2 127 PRT PartialMycobacterium xenopi GyrA 2 Asp Arg Ser His Ala Lys Ser Ala Arg Ser ValAla Glu Thr Met Gly 1 5 10 15 Asn Tyr His Pro His Gly Asp Ala Ser IleTyr Asp Thr Leu Val Arg 20 25 30 Met Ala Gln Pro Trp Ser Met Arg Tyr ProLeu Val Asp Gly Gln Gly 35 40 45 Asn Phe Gly Ser Pro Gly Asn Asp Pro ProAla Ala Met Arg Tyr Thr 50 55 60 Glu Ala Pro Leu Thr Pro Leu Ala Met GluMet Leu Arg Glu Ile Asp 65 70 75 80 Glu Glu Thr Val Asp Phe Ile Pro AsnTyr Asp Gly Arg Val Gln Glu 85 90 95 Pro Thr Val Leu Pro Ser Arg Phe ProAsn Leu Leu Ala Asn Gly Ser 100 105 110 Gly Gly Ile Ala Val Gly Met AlaThr Asn Ile Pro Pro His Asn 115 120 125 3 438 PRT Escherichia coli DnaB3 Pro Pro His Ser Ile Glu Ala Glu Gln Ser Val Leu Gly Gly Leu Met 1 5 1015 Leu Asp Asn Glu Arg Trp Asp Asp Val Ala Glu Arg Val Val Ala Asp 20 2530 Asp Phe Tyr Thr Arg Pro His Arg His Ile Phe Thr Glu Met Ala Arg 35 4045 Leu Gln Glu Ser Gly Ser Pro Ile Asp Leu Ile Thr Leu Ala Glu Ser 50 5560 Leu Glu Arg Gln Gly Gln Leu Asp Ser Val Gly Gly Phe Ala Tyr Leu 65 7075 80 Ala Glu Leu Ser Lys Asn Thr Pro Ser Ala Ala Asn Ile Ser Ala Tyr 8590 95 Ala Asp Ile Val Arg Glu Arg Ala Val Val Arg Glu Met Ile Ser Val100 105 110 Ala Asn Glu Ile Ala Glu Ala Gly Phe Asp Pro Gln Gly Arg ThrSer 115 120 125 Glu Asp Leu Leu Asp Leu Ala Glu Ser Arg Val Phe Lys IleAla Glu 130 135 140 Ser Arg Ala Asn Lys Asp Glu Gly Pro Lys Asn Ile AlaAsp Val Leu 145 150 155 160 Asp Ala Thr Val Ala Arg Ile Glu Gln Leu PheGln Gln Pro His Asp 165 170 175 Gly Val Thr Gly Val Asn Thr Gly Tyr AspAsp Leu Asn Lys Lys Thr 180 185 190 Ala Gly Leu Gln Pro Ser Asp Leu IleIle Val Ala Ala Arg Pro Ser 195 200 205 Met Gly Lys Thr Thr Phe Ala MetAsn Leu Val Glu Asn Ala Ala Met 210 215 220 Leu Gln Asp Lys Pro Val LeuIle Phe Ser Leu Glu Met Pro Ser Glu 225 230 235 240 Gln Ile Met Met ArgSer Leu Ala Ser Leu Ser Arg Val Asp Gln Thr 245 250 255 Lys Ile Arg ThrGly Gln Leu Asp Asp Glu Asp Trp Ala Arg Ile Ser 260 265 270 Gly Thr MetGly Ile Leu Leu Glu Lys Arg Asn Ile Tyr Ile Asp Asp 275 280 285 Ser SerGly Leu Thr Pro Thr Glu Val Arg Ser Arg Ala Arg Arg Ile 290 295 300 AlaArg Glu His Gly Gly Ile Gly Leu Ile Met Ile Asp Tyr Leu Gln 305 310 315320 Leu Met Arg Val Pro Ala Leu Ser Asp Asn Arg Thr Leu Glu Ile Ala 325330 335 Glu Ile Ser Arg Ser Leu Lys Ala Leu Ala Lys Glu Leu Asn Val Pro340 345 350 Val Val Ala Leu Ser Gln Leu Asn Arg Ser Leu Glu Gln Arg AlaAsp 355 360 365 Lys Arg Pro Val Asn Ser Asp Leu Arg Glu Ser Gly Ser IleGlu Gln 370 375 380 Asp Ala Asp Leu Ile Met Phe Ile Tyr Arg Asp Glu ValTyr His Glu 385 390 395 400 Asn Ser Asp Leu Lys Gly Ile Ala Glu Ile IleIle Gly Lys Gln Arg 405 410 415 Asn Gly Pro Ile Gly Thr Val Arg Leu ThrPhe Asn Gly Gln Trp Ser 420 425 430 Arg Phe Asp Asn Tyr Ala 435 4 434PRT Partial Mycobacterium tuberculosis DnaB 4 Pro Pro Gln Asp Leu AlaAla Glu Gln Ser Val Leu Gly Gly Met Leu 1 5 10 15 Leu Ser Lys Asp AlaIle Ala Asp Val Leu Glu Arg Leu Arg Pro Gly 20 25 30 Asp Phe Tyr Arg ProAla His Gln Asn Val Tyr Asp Ala Ile Leu Asp 35 40 45 Leu Tyr Gly Arg GlyGlu Pro Ala Asp Ala Val Thr Val Ala Ala Glu 50 55 60 Leu Asp Arg Arg GlyLeu Leu Arg Arg Ile Gly Gly Ala Pro Tyr Leu 65 70 75 80 His Thr Leu IleSer Thr Val Pro Thr Ala Ala Asn Ala Gly Tyr Tyr 85 90 95 Ala Ser Ile ValAla Glu Lys Ala Leu Leu Arg Arg Leu Val Glu Ala 100 105 110 Gly Thr ArgVal Val Gln Tyr Gly Tyr Ala Gly Ala Glu Gly Ala Asp 115 120 125 Val AlaGlu Val Val Asp Arg Ala Gln Ala Glu Ile Tyr Asp Val Ala 130 135 140 AspArg Arg Leu Ser Glu Asp Phe Val Ala Leu Glu Asp Leu Leu Gln 145 150 155160 Pro Thr Met Asp Glu Ile Asp Ala Ile Ala Ser Ser Gly Gly Leu Ala 165170 175 Arg Gly Val Ala Thr Gly Phe Thr Glu Leu Asp Glu Val Thr Asn Gly180 185 190 Leu His Pro Gly Gln Met Val Ile Val Ala Ala Arg Pro Gly ValGly 195 200 205 Lys Ser Thr Leu Gly Leu Asp Phe Met Arg Ser Cys Ser IleArg His 210 215 220 Arg Met Ala Ser Val Ile Phe Ser Leu Glu Met Ser LysSer Glu Ile 225 230 235 240 Val Met Arg Leu Leu Ser Ala Glu Ala Lys IleLys Leu Ser Asp Met 245 250 255 Arg Ser Gly Arg Met Ser Asp Asp Asp TrpThr Arg Leu Ala Arg Arg 260 265 270 Met Ser Glu Ile Ser Glu Ala Pro LeuPhe Ile Asp Asp Ser Pro Asn 275 280 285 Leu Thr Met Met Glu Ile Arg AlaLys Ala Arg Arg Leu Arg Gln Lys 290 295 300 Ala Asn Leu Lys Leu Ile ValVal Asp Tyr Leu Gln Leu Met Thr Ser 305 310 315 320 Gly Lys Lys Tyr GluSer Arg Gln Val Glu Val Ser Glu Phe Ser Arg 325 330 335 His Leu Lys LeuLeu Ala Lys Glu Leu Glu Val Pro Val Val Ala Ile 340 345 350 Ser Gln LeuAsn Arg Gly Pro Glu Gln Arg Thr Asp Lys Lys Pro Met 355 360 365 Leu AlaAsp Leu Arg Glu Ser Gly Ser Leu Glu Gln Asp Ala Asp Val 370 375 380 ValIle Leu Leu His Arg Pro Asp Ala Phe Asp Arg Asp Asp Pro Arg 385 390 395400 Gly Gly Glu Ala Asp Phe Ile Leu Ala Lys His Arg Asn Gly Pro Thr 405410 415 Lys Thr Val Thr Val Ala His Gln Leu His Leu Ser Arg Phe Ala Asn420 425 430 Met Ala 5 7 PRT Mycobacterium tuberculosis DnaB 5 Thr ValGln Ser Thr Lys Arg 1 5 6 7 PRT Mycobacterium tuberculosis DnaB 6 ArgPro Ala Pro Arg Pro Leu 1 5 7 7 PRT Mycobacterium tuberculosis DnaB 7Pro Thr Ala Arg Thr Tyr Glu 1 5 8 12 PRT Mycobacterium tuberculosis DnaB8 Pro Thr Arg Pro Thr Ala Pro Pro Leu Asn Phe Ser 1 5 10 9 12 PRTMycobacterium tuberculosis DnaB 9 His Pro Asn Pro His Pro Thr Leu SerGly Gln Arg 1 5 10 10 7 PRT Mycobacterium tuberculosis DnaB 10 Asp LeuPro Met Val Glu Glu 1 5 11 198 PRT Mycobacterium xenopi Gyrase A intein11 Cys Ile Thr Gly Asp Ala Leu Val Ala Leu Pro Glu Gly Glu Ser Val 1 510 15 Arg Ile Ala Asp Ile Val Pro Gly Ala Arg Pro Asn Ser Asp Asn Ala 2025 30 Ile Asp Leu Lys Val Leu Asp Arg His Gly Asn Pro Val Leu Ala Asp 3540 45 Arg Leu Phe His Ser Gly Glu His Pro Val Tyr Thr Val Arg Thr Val 5055 60 Glu Gly Leu Arg Val Thr Gly Thr Ala Asn His Pro Leu Leu Cys Leu 6570 75 80 Val Asp Val Ala Gly Val Pro Thr Leu Leu Trp Lys Leu Ile Asp Glu85 90 95 Ile Lys Pro Gly Asp Tyr Ala Val Ile Gln Arg Ser Ala Phe Ser Val100 105 110 Asp Cys Ala Gly Phe Ala Arg Gly Lys Pro Glu Phe Ala Pro ThrThr 115 120 125 Tyr Thr Val Gly Val Pro Gly Leu Val Arg Phe Leu Glu AlaHis His 130 135 140 Arg Asp Pro Asp Ala Gln Ala Ile Ala Asp Glu Leu ThrAsp Gly Arg 145 150 155 160 Phe Tyr Tyr Ala Lys Val Ala Ser Val Thr AspAla Gly Val Gln Pro 165 170 175 Val Tyr Ser Leu Arg Val Asp Thr Ala AspHis Ala Phe Ile Thr Asn 180 185 190 Gly Phe Val Ser His Asn 195 12 85PRT Gallus gallus alpha-spectrin fragment 12 Met Arg Asn Thr Thr Gly ValThr Glu Glu Ala Leu Lys Glu Phe Ser 1 5 10 15 Met Met Phe Lys His PheAsp Lys Asp Lys Ser Gly Arg Leu Asn His 20 25 30 Gln Glu Phe Lys Ser CysLeu Arg Ser Leu Gly Tyr Asp Leu Pro Met 35 40 45 Val Glu Glu Gly Glu ProAsp Pro Glu Phe Glu Ser Ile Leu Asp Thr 50 55 60 Val Asp Pro Asn Arg AspGly His Val Ser Leu Gln Glu Tyr Met Ala 65 70 75 80 Phe Met Ile Ser Arg85 13 416 PRT Mycobacterium tuberculosis DnaB intein 13 Cys Leu Thr AlaSer Thr Arg Ile Leu Arg Ala Asp Thr Gly Ala Glu 1 5 10 15 Val Ala PheGly Glu Leu Met Arg Ser Gly Glu Arg Pro Met Val Trp 20 25 30 Ser Leu AspGlu Arg Leu Arg Met Val Ala Arg Pro Met Ile Asn Val 35 40 45 Phe Pro SerGly Arg Lys Glu Val Phe Arg Leu Arg Leu Ala Ser Gly 50 55 60 Arg Glu ValGlu Ala Thr Gly Ser His Pro Phe Met Lys Phe Glu Gly 65 70 75 80 Trp ThrPro Leu Ala Gln Leu Lys Val Gly Asp Arg Ile Ala Ala Pro 85 90 95 Arg ArgVal Pro Glu Pro Ile Asp Thr Gln Arg Met Pro Glu Ser Glu 100 105 110 LeuIle Ser Leu Ala Arg Met Ile Gly Asp Gly Ser Cys Leu Lys Asn 115 120 125Gln Pro Ile Arg Tyr Glu Pro Val Asp Glu Ala Asn Leu Ala Ala Val 130 135140 Thr Val Ser Ala Ala His Ser Asp Arg Ala Ala Ile Arg Asp Asp Tyr 145150 155 160 Leu Ala Ala Arg Val Pro Ser Leu Arg Pro Ala Arg Gln Arg LeuPro 165 170 175 Arg Gly Arg Cys Thr Pro Ile Ala Ala Trp Leu Ala Gly LeuGly Leu 180 185 190 Phe Thr Lys Arg Ser His Glu Lys Cys Val Pro Glu AlaVal Phe Arg 195 200 205 Ala Pro Asn Asp Gln Val Ala Leu Phe Leu Arg HisLeu Trp Ser Ala 210 215 220 Gly Gly Ser Val Arg Trp Asp Pro Thr Asn GlyGln Gly Arg Val Tyr 225 230 235 240 Tyr Gly Ser Thr Ser Arg Arg Leu IleAsp Asp Val Ala Gln Leu Leu 245 250 255 Leu Arg Val Gly Ile Phe Ser TrpIle Thr His Ala Pro Lys Leu Gly 260 265 270 Gly His Asp Ser Trp Arg LeuHis Ile His Gly Ala Lys Asp Gln Val 275 280 285 Arg Phe Leu Arg His ValGly Val His Gly Ala Glu Ala Val Ala Ala 290 295 300 Gln Glu Met Leu ArgGln Leu Lys Gly Pro Val Arg Asn Pro Asn Leu 305 310 315 320 Asp Ser AlaPro Lys Lys Val Trp Ala Gln Val Arg Asn Arg Leu Ser 325 330 335 Ala LysGln Met Met Asp Ile Gln Leu His Glu Pro Thr Met Trp Lys 340 345 350 HisSer Pro Ser Arg Ser Arg Pro His Arg Ala Glu Ala Arg Ile Glu 355 360 365Asp Arg Ala Ile His Glu Leu Ala Arg Gly Asp Ala Tyr Trp Asp Thr 370 375380 Val Val Glu Ile Thr Ser Ile Gly Asp Gln His Val Phe Asp Gly Thr 385390 395 400 Val Ser Gly Thr His Asn Phe Val Ala Asn Gly Ile Ser Leu HisAsn 405 410 415 14 8 PRT Mycobacterium xenopi Gyrase A 14 Asp Ser AlaAla Ala Met Arg Tyr 1 5 15 31 DNA Escherichia coli Gyrase A 15gataggctag cgatgagcga ccttgcgaga g 31 16 32 DNA Escherichia coli GyraseA 16 tgaagcaatt gaattattct tcttctggct cg 32 17 32 DNA Nocardiaotitidis-caviarum 17 cggcgactct gcggccgcaa tgcgttatac gg 32 18 32 DNANocardia otitidis-caviarum 18 ccgtataacg cattgcggcc gcagagtcgc cg 32 1931 DNA Xanthomonas badrii 19 gaactgatgg ccgctctaga aaaagagacg g 31 20 31DNA Xanthomonas badrii 20 ccgtctcttt ttctagagcg gccatcagtt c 31 21 61DNA Bacillus lentus 21 ggccgcaatg cgttatacgg aaatccgctt agcgaaaattgcccatgaac tgatggccga 60 t 61 22 60 DNA Bacillus lentus 22 ctagatcggcatcagttcat gggcaatttt cgctaagcgg atttccgtat aacgcattgc 60 23 39 DNAMycobacterium xenopi Gyrase A 23 cgacccgcgc ggccgcaatg cgttattgcatcacgggag 39 24 45 DNA Mycobacterium xenopi 24 gccaaaggcg ctaagcggatttccgtgttg tggctgacga acccg 45 25 31 DNA Streptomyces phaeochromogenes25 atgggcatgc atatatatat aggcctgggc c 31 26 30 DNA Streptomycesphaeochromogenes 26 caggcctata tatatatgca tgcccattcg 30 27 39 DNAStreptomyces griseoruber 27 gtttaagtct tgcttgcgat cgcttggcta tgacctgcc39 28 38 DNA Streptomyces griseoruber 28 gcctgacccc gaatttgaatcgattcttga cactgttg 38 29 38 DNA Caryophanon latum 29 gcctgaccccgaatttgaat cgattcttga cactgttg 38 30 38 DNA Caryophanon latum 30caacagtgtc aagaatcgat tcaaattcgg ggtcaggc 38 31 34 DNA Gallus gallusalpha-spectrin 31 aatggtgcat gcaaggagat ggcgcccaac agtc 34 32 41 DNAGallus gallus alpha-spectrin 32 gctttggcta gctttcctgt gtcacctgctgatcatgaac g 41 33 29 DNA Proteus vulgaris 33 gcgtaaagct cgcgaccgtgctcatatcc 29 34 29 DNA Proteus vulgaris 34 ggatatgagc acggtcgcgagctttacgc 29 35 53 DNA Gallus gallus alpha-spectrin ((W)NN)7-12 =synthetic randon oligo 35 tgtcaagaat cgattcaaat tcggggtcag gctctccwnnatagccaagc gat 53 36 11 DNA Gallus gallus alpha-spectrin 36 cgcttggcta t11 37 31 DNA Mycobacterium tuberculosis 37 aggtgagaat tcatggcggtcgttgatgac c 31 38 36 DNA Mycobacterium tuberculosis 38 tatataaagctttcatgtca ccgagccatg ttggcg 36 39 31 DNA Mycobacterium tuberculosis 39aggtgagaat tcatggcggt cgttgatgac c 31 40 33 DNA Mycobacteriumtuberculosis 40 tttcccacgc ccgggcacgc cgccacgatg acc 33 41 32 DNAAcetobacter aceti 41 gccgccgatc cgcgacatcg tagatttcgg cc 32 42 32 DNAAcetobacter aceti 42 ggccgaaatc tacgatgtcg cggatcggcg gc 32 43 89 DNAGallus gallus alpha-spectrin ((W)NN)7-12 = synthetic randon oligo 43tgtcaagaat cgattcaaat tcggggtcag gctctccwnn atagccaagc gatcgcaggc 60agcttttaaa gccctgatgg ttcagacgt 89 44 15 DNA Gallus gallusalpha-spectrin 44 ctgaaccatc agggc 15 45 7 PRT Mycobacterium xenopiGyrase A 45 Glu Ile Arg Leu Ala Lys Ile 1 5 46 199 PRT Mycobacteriumxenopi Gyrase A 46 Cys Ile Thr Gly Asp Ala Leu Val Ala Leu Pro Glu GlyGlu Ser Val 1 5 10 15 Arg Ile Ala Asp Ile Val Pro Gly Ala Arg Pro AsnSer Asp Asn Ala 20 25 30 Ile Asp Leu Lys Val Leu Asp Arg His Gly Asn ProVal Leu Ala Asp 35 40 45 Arg Leu Phe His Ser Gly Glu His Pro Val Tyr ThrVal Arg Thr Val 50 55 60 Glu Gly Leu Arg Val Thr Gly Thr Ala Asn His ProLeu Leu Cys Leu 65 70 75 80 Val Asp Val Ala Gly Val Pro Thr Leu Leu TrpLys Leu Ile Asp Glu 85 90 95 Ile Lys Pro Gly Asp Tyr Ala Val Ile Gln ArgSer Ala Phe Ser Val 100 105 110 Asp Cys Ala Gly Phe Ala Arg Gly Lys ProGlu Phe Ala Pro Thr Thr 115 120 125 Tyr Thr Val Gly Val Pro Gly Leu ValArg Phe Leu Glu Ala His His 130 135 140 Arg Asp Pro Asp Ala Gln Ala IleAla Asp Glu Leu Thr Asp Gly Arg 145 150 155 160 Phe Tyr Tyr Ala Lys ValAla Ser Val Thr Asp Ala Gly Val Gln Pro 165 170 175 Val Tyr Ser Leu ArgVal Asp Thr Ala Asp His Ala Phe Ile Thr Asn 180 185 190 Gly Phe Val SerHis Asn Thr 195

What is claimed is:
 1. A positive genetic selection system employing aprecursor comprising a native intein in its natural or homologousmodifiable extein context for the screening of agents which inhibit oractivate protein splicing, said selection system comprising: (1) a hostcell which contains a first gene encoding a non-selectable form of atarget enzyme, and (2) a second gene encoding a selectable form of saidtarget enzyme which is dominantly cytotoxic upon interaction underpredetermined selection conditions, said second gene containing anintein, wherein the inhibition or activation of said selectable form ofsaid target enzyme by a given agent affects the viability of said hostcell.
 2. The positive genetic selection system of claim 1, wherein theactivation of said selectable form of said target enzyme by a givenagent results in the death of the host cell.
 3. The positive geneticselection system of claim 1, wherein the inhibition of said selectableform of said target enzyme by a given agent results in the viability ofthe host cell.
 4. The positive genetic selection system of claim 1,wherein said host cell contains a first gene encoding a drug-resistantform of the target enzyme and a second gene encoding a drug-sensitiveform of the target enzyme which is dominantly cytotoxic upon interactionwith said drug.
 5. The positive genetic selection system of claim 1,wherein said first gene encodes a wild type form of said target enzymeand said second gene encodes a dominant cytotoxic form of said targetenzyme.
 6. The positive genetic selection system of claim 4, wherein inthe absence of a silent mutation of said extein, said intein is selectedfrom the group consisting of an intein inserted into the drug-sensitiveform of said target enzyme and a natural intein in a mutated native orhomologous extein, wherein said mutation renders the extein cytotoxicupon interaction with said drug.
 7. The positive genetic selectionsystem of claim 1, wherein said agent comprises an in vivo peptidelibrary or derivatives thereof.
 8. The positive genetic selection systemof claim 4, wherein said intein comprises the M. xenopi GyrA intein,said homologous extein comprises E.coli GyrA and said host cellcomprises E.coli.
 9. The positive genetic selection system of claim 8,wherein said drug-resistant form of said target enzyme is the Ser83mutant of E.coli GyrA.
 10. The positive genetic selection system ofclaim 7, wherein said peptide library comprises a combinatorial peptidelibrary in a fragment of chicken α-spectrin.
 11. The positive geneticselection system of claim 5, wherein in the absence of a silent mutationin said extein, said intein is selected from the group consisting of anintein inserted in to the cytotoxic form of said target enzyme and anatural intein in a mutated native or homologous extein, wherein saidmutated native extein is cytotoxic.
 12. The positive genetic selectionsystem of claim 11, wherein said agent comprises an in vivo peptidelibrary or derivatives thereof.
 13. The positive genetic selectionsystem of claim 11, wherein said natural intein is the M. tuberculosisDnaB intein and said mutated native extein is the M.tuberculosis R231Cmutant, and wherein said host cell is E. coli.
 14. The positive geneticselection system of claim 13, wherein said in vivo peptide librarycomprises a combinatorial peptide library in a fragment of chickenα-spectrin.
 15. A method of screening for agents which inhibit proteinsplicing, said method comprising the steps of: (a) creating a positiveselection system comprising a host cell containing a gene encoding adominantly cytotoxic protein containing an intein; and (b) culturing thehost cell of step (a) in the presence of test agents, wherein theinhibition of splicing of said cytotoxic protein results in viability ofsaid host cell.
 16. The method of claim 15, wherein said agent isexpressed within the host cell as a protein or portion thereof and saidagent is identified by the gene encoding said agent from said survivinghost.
 17. The method of claim 15, wherein said positive selection systemof step (a) comprises the positive genetic selection wherein said hostcell contains a first gene encoding a drug-resistant form of the targetenzyme and a second gene encoding a drug-sensitive form of the targetenzyme which is dominantly cytotoxic upon interaction with said drug,and wherein step (b) further comprises culturing said host cell of step(a) in the presence of said drug.
 18. The method of claim 15, whereinsaid positive selection system of step (a) comprises a first geneencoding a wild type form of said target enzyme and a second geneencoding a dominant cytotoxic form of said target enzyme.
 19. The methodof claims 17 or 18, wherein said host cell of step (a) expresses an invivo peptide library or derivatives thereof, and wherein said testagents of step (b) comprise said in vivo peptide library or derivativesthereof.
 20. The method of claim 19 wherein said in vivo peptide librarycomprises a combinatorial peptide library in a fragment of chickenα-spectrin.
 21. A method for screening of agents which activate proteinsplicing, said method comprising the steps of: (a) creating a positiveselection system comprising a host cell containing a gene encoding adominantly cytotoxic protein, said gene containing an inactive intein;and (b) culturing said host cell of step (a) in the presence ofindividual test agents, wherein the activation of splicing of saidcytotoxic protein results in host cell death.
 22. The method of claim21, wherein said agent is controllably expressed within the host cell asa protein or portion thereof and said agent is identified by theidentification of the gene encoding said agent in a parallel sample ofsaid host cell in which expression of said agent was not activated. 23.The method of claim 21, wherein said positive selection system of step(a) comprises the positive genetic selection system wherein said hostcell contains a first gene encoding a drug-resistant form of the targetenzyme and a second gene encoding a drug-sensitive form of the targetenzyme which is dominantly cytotoxic upon interaction with said drug,and wherein step (b) further comprises culturing said positive selectionsystem of step (a) in the presence of said drug.
 24. The method of claim21, wherein said positive selection system of step (a) comprises thepositive genetic selection system wherein the drug-resistant form ofsaid target enzyme is the Ser83 mutant of E.coli GyrA.
 25. The method ofclaims 21 or 22, wherein said host cell of step (a) expresses an in vivopeptide library or derivatives thereof, and wherein said test agents ofstep (b) comprise said in vivo peptide library or derivatives thereof.26. The method of claim 25, wherein said in vivo peptide librarycomprises a combinatorial peptide library in a fragment of chickenα-spectrin.
 27. A positive genetic selection system for the screening ofagents which inhibit protein splicing, said selection system comprising:(1) a host cell which contains a first gene encoding a controllable formof a target enzyme which is required for cell viability, and (2) asecond gene encoding an expressed form of said target enzyme, saidsecond gene containing an intein, wherein the inhibition of splicing ofsaid target enzyme by a given agent results in the reduced viability ordeath of said host cell under conditions which do not permit theexpression of said controllable first gene of said target enzyme. 28.The selection system of claim 27, wherein said controllable form of saidtarget protein is selected from the group consisting of a drug-sensitivetarget protein, an inducer-sensitive target protein, atemperature-sensitive target protein, and a target protein operablylinked to a controllable promoter.
 29. The selection system of claim 27,wherein said intein is selected from the group consisting of a foreignintein inserted into the homologous extein of said target enzyme and anatural intein in a native extein.
 30. A positive genetic selectionsystem for the screening of agents which activate protein splicing, saidselection system comprising: (1) a host cell which contains a first geneencoding a controllable form of a target enzyme required for cellviability, and (2) a second gene encoding an expressed form of saidtarget enzyme, said second gene containing an inactive intein, whereinthe activation of splicing of said target enzyme by a given agentresults in the survival of said host cell under conditions which do notpermit the expression of said controllable first gene of said targetenzyme.
 31. The selection system of claim 30, wherein said controllabletarget protein is selected from the group consisting of a drug-sensitivetarget protein, an inducer-sensitive target protein, atemperature-sensitive target protein, and a target protein operablylinked to a controllable promoter.
 32. The selection system of claim 30,wherein said inactive intein is selected from the group consisting of aforeign inactive intein inserted into the homologous extein of saidtarget enzyme and a natural inactive intein in a native extein.
 33. Themethod of claims 27 or 30, wherein said host cell of step (a) expressesan in vivo peptide library or derivatives thereof, and wherein said testagents of step (b) comprise said in vivo peptide library or derivativesthereof.
 34. The method of claim 33, wherein said in vivo peptidelibrary comprises a combinatorial peptide library in a fragment ofchicken α-spectrin.
 35. A method of screening for agents which inhibitprotein splicing, said method comprising the steps of: (a) culturing thepositive selection system of claim 27 in the presence of test agentsunder non-permissive conditions; and (b) identifying non-surviving hostcells from step (a), wherein said agent inhibits protein splicing. 36.The method of claim 35, wherein said agent is expressed within the hostcell as a protein or portion thereof and wherein said non-surviving hostcells of step (b) contain test agents which inhibit protein splicing.37. A method of screening for agents which activate protein splicing,said method comprising the steps of: (a) culturing the positiveselection system of claim 30 in the presence of test agents undernon-permissive conditions; and (b) identifying surviving host cells fromstep (a), wherein said agent activates protein splicing.
 38. The methodof claim 37, wherein said agent is expressed within the host cell as aprotein or portion thereof and wherein said surviving host cells of step(b) contain test agents which activate protein splicing.
 39. The methodof claims 36 or 38, wherein said host cell of step (a) expresses an invivo peptide library or derivatives thereof, and wherein said testagents of step (a) comprise said in vivo peptide library or derivativesthereof.
 40. The method of claim 39, wherein said in vivo peptidelibrary comprises a combinatorial peptide library in a fragment ofchicken α-spectrin.
 41. A reporter system for the screening of agentswhich inhibit protein splicing, said reporter system comprising a hostcell which contains a reporter gene encoding a non-essential protein,said reporter gene containing an intein, wherein said intein is selectedfrom the group consisting of a foreign intein inserted into thehomologous or non-homologous extein of said reporter gene in the absenceof a silent mutation of said extein and a natural intein in a nativereporter extein, and wherein the inhibition of splicing of saidnon-essential protein by a given agent results in a specific detectablephenotype of said host cell.
 42. A reporter system for the screening ofagents which activate protein splicing, said reporter system comprisinga host cell which contains a reporter gene encoding a non-essentialprotein, said reporter gene containing an inactive intein, wherein saidinactive intein is selected from the group consisting of a foreigninactive intein inserted into the homologous or non-homologous extein ofsaid reporter gene in the absence of a silent mutation of said exteinand a natural inactive intein in a native reporter extein, and whereinthe activation of splicing of said non-essential protein by a givenagent results in a specific selectable phenotype of said host cell. 43.The method of screening for agents which inhibit protein splicing, saidmethod comprising the steps of: (a) culturing the reporter system ofclaim 41 in the presence of test agents; and (b) identifying host cellsfrom step (a) having a specific detectable phenotype, wherein said hostcells with detectable phenotype are in the presence of test agents whichinhibit protein splicing.
 44. The method of claim 43 wherein said agentis expressed within the host cell as a protein or portion thereof andwherein step (b) comprises identifying host cells from step (a) having aspecific detectable phenotype, wherein said host cells with detectablephenotype contain test agents which inhibit protein splicing.
 45. Amethod of screening for agents which activate protein splicing, saidmethod comprising the steps of: (a) culturing the reporter system ofclaim 42 in the presence of test agents; and (b) identifying host cellsfrom step (a) having a specific detectable phenotype, wherein said hostcells with detectable phenotype are in the presence of test agents whichactivate protein splicing.
 46. The method of claim 45 wherein said agentis expressed within the host cell as a protein or portion thereof andwherein step (b) comprises identifying host cells from step (a) having aspecific detectable phenotype, wherein said host cells with detectablephenotype contain test agents which activate protein splicing.
 47. Themethod of claims 44 or 46, wherein said host cell of step (a) expressesan in vivo peptide library or derivatives thereof, and wherein said testagents of step (a) comprise said in vivo peptide library or derivativesthereof.
 48. The method of claim 47, wherein said in vivo peptidelibrary comprises a combinatorial peptide library in a fragment ofchicken α-spectrin.
 49. A method of controlling gene expression in vivo,said method comprising the steps of: (a) replacing a homologous exteingene in a host cell with a gene containing an intein; and (b) modulatingthe splicing of the intein-containing gene of step (a) with agents whichinhibit or activate said splicing.
 50. The method of claim 49, whereinsaid replacement of step (a) comprises inserting said intein gene intosaid homologous extein gene by homologous recombination.
 51. The methodof claim 49, wherein said replacement of step (a) comprises inactivatingsaid homologous extein gene and said intein-containing gene isintroduced as a second gene in the cell.
 52. A method of controlling thedelivery of a drug that is at least partially proteinaceous in vivo,said method comprising the steps of: (a) inserting an intein into theprotein portion of said drug to create an inactive drug; (b)administering said inactive drug of step (a); and (c) activating proteinsplicing of said inactive drug to produce an active drug.
 53. The methodof claim 52, wherein step (b) further comprises utilizing gene therapyto target said inactive drug to a desired tissue.
 54. The method ofclaim 52, wherein said intein of step (a) comprises atemperature-sensitive intein, and wherein said activation of step (c)comprises exposing a desired target tissue to any treatment whichincreases or decreases temperature of said target tissue thus inducingsplicing of said temperature-sensitive intein.
 55. The method of claim52, wherein said activation of step (c) comprises injecting a desiredtarget tissue with an agent which activates splicing.
 56. The method ofclaim 52, wherein step (a) further comprises utilizing gene therapy totarget an agent which activates splicing to a desired tissue, andwherein said administration of step (b) comprises systemicadministration.
 57. The method of claim 52, wherein said administrationof step (b) comprises systemic transformation with a non-cell specificvector containing said inactive drug of step (a) operably linked to adesired tissue-specific promoter, and wherein said inactive drug isexpressed only in cells which can activate said tissue-specificpromoter.
 58. A method for generating temperature sensitive mutants ofthe Mxe GyrA intein in E.coli GyrA, said method comprising the steps of:(a) identification of the region containing the Mxe GyrA inteinbeta-strand B8 and the loop between Beta-strands B8 and B9 in E.coliGyrA; (b) mutating said region; and (c) introducing the mutated inteingene into a cell and examining the ability to splice under permissiveand non-permissive temperatures.
 59. A method for generating temperaturesensitive mutants of an intein, said method comprising the steps of: (a)identification of a region homologous to the Mxe GyrA intein beta-strandB8 and the loop between beta-strands B8 and B9 in an intein; (b)mutating said homologous region of said second intein; and (c)introducing the mutated intein gene into a cell and examining theability to splice under permissive and non-permissive temperatures. 60.A method of screening for temperature-sensitive inteins, said methodcomprising the steps of (a) creating a positive selection systemcomprising a host cell containing a gene encoding a dominantly cytotoxicprotein, said gene containing an intein, and wherein said intein ismutagenized; and (b) culturing the host cell of step (a) at a range oftemperatures, wherein at a predetermined temperature, the protein failsto splice and results in viability of said host cell.
 61. A method ofscreening for temperature-sensitive inteins, said method comprising thesteps of (a) creating a positive selection system comprising a host cellcontaining a gene encoding a dominantly cytotoxic protein, said genecontaining an inactive intein, and wherein said intein is mutagenized;and (b) culturing the host cell of step (a) at a range of temperatures,wherein at a predetermined temperature, the intein splices and resultsin the death of the host cell.
 62. A positive selection system for thescreening of temperatures which inhibit protein splicing intemperature-sensitive inteins, said selection system comprising (1) ahost cell which contains a first gene encoding a controllable form of atarget enzyme which is required for cell viability, and (2) a secondgene encoding an expressed form of said target enzyme, said second genecontaining an intein, wherein said intein is mutagenized, and whereinthe inhibition of splicing of said target enzyme by a givennon-permissive temperature results in the reduced viability or death ofsaid host cell under conditions which do not permit the expression ofsaid controllable first gene of said target enzyme.
 63. A positiveselection system for the screening of temperatures which activateprotein splicing in temperature-sensitive inteins, said selection systemcomprising (1) a host cell which contains a first gene encoding acontrollable form of a target enzyme which is required for cellviability, and (2) a second gene encoding an expressed form of saidtarget enzyme, said second gene containing an intein, wherein saidintein is mutagenized, and wherein the activation of splicing of saidtarget enzyme by a given permissive temperature results in the viabilityof said host cell under conditions which do not permit the expression ofsaid controllable first gene of said target enzyme.
 64. A method ofscreening for temperatures which inhibit protein splicing intemperature-sensitive inteins, said method comprising the steps of: (a)culturing the positive selection system of claim 62 in the presence ofnon-permissive temperatures; and (b) identifying non-surviving hostcells from step (a), wherein said temperature inhibits protein splicing.65. A method of screening for temperatures which activate proteinsplicing in temperature-sensitive inteins, said method comprising thesteps of: (a) culturing the positive selection system of claim 63 in thepresence of permissive temperatures; and (b) identifying surviving hostcells from step (a), wherein said temperature activates proteinsplicing.
 66. A reporter system for the screening of temperatures whichinhibit protein splicing in temperature-sensitive inteins, said reportersystem comprising a host cell which contains a reporter gene encoding anon-essential protein, said reporter gene containing an intein, whereinsaid intein is selected from the group consisting of a foreign inteininserted into the homologous or non-homologous extein of said reportergene in the absence of a silent mutation of said extein and a naturalintein in a native reporter extein, and wherein said intein ismutagenized, and wherein the inhibition of splicing of saidnon-essential protein by a non-permissive temperature results in aspecific detectable phenotype.
 67. A reporter system for the screeningof temperatures which activate protein splicing in temperature-sensitiveinteins, said reporter system comprising a host cell which contains areporter gene encoding a non-essential protein, said reporter genecontaining an intein, wherein said intein is selected from the groupconsisting of a foreign intein inserted into the homologous ornon-homologous extein of said reporter gene in the absence of a silentmutation of said extein and a natural intein in a native reporterextein, and wherein said intein is mutagenized, and wherein theactivation of splicing of said non-essential protein by a permissivetemperature results in a specific detectable phenotype.
 68. A method forscreening for temperatures which inhibit protein splicing intemperature-sensitive inteins, said method comprising the steps of: (a)culturing the reporter system of claim 66 in the presence of a range oftemperatures; and (b) identifying host cells from step (a) having aspecific detectable phenotype, wherein said host cells with detectablephenotype are in the presence non-permissive temperatures which inhibitprotein splicing.
 69. A method for screening for temperatures whichactivate protein splicing in temperature-sensitive inteins, said methodcomprising the steps of: (a) culturing the reporter system of claim 67in the presence of a range of temperatures; and (b) identifying hostcells from step (a) having a specific detectable phenotype, wherein saidhost cells with detectable phenotype are in the presence permissivetemperatures which activate protein splicing.
 70. The positive geneticselection system of claim 6, 11, 29 or 32, wherein the extein is aheterologous extein and the intein is inserted into the extein at a sitewhich is substantially identical to the homologous extein from about oneto about five amino acid residues at either or both ends of the intein.71. The reporter system of claim 41, wherein the extein is aheterologous extein and the intein is inserted into the extein at a sitewhich is substantially identical to the homologous extein from about oneto five amino acid residues at either or both ends of the intein. 72.The positive selection system of claim 6, 11, 29 or 32, wherein theextein is a heterologous extein and one to five amino acid residues ofthe native extein are present at one or both ends of the intein and saidone to five amino acid residues are inserted into the heterologousextein along with the intein.
 73. The reporter system of claim 41,wherein the extein is a heterologous extein and one to five amino acidresidues of the native extein are present at one or both ends of theintein and said one to five amino acid residues are inserted into theheterologous extein along with the intein.
 74. The positive geneticselection system of claims 6, 11, 29 or 32, wherein the insertion siteis selected from the group consisting essentially of a surface locationon the extein, a loop region of the extein, a protease sensitive sitewithin the extein, or at a position known to permit insertion of one ormore amino acid residues in the extein without inactivating the extein.75. The positive genetic selection system of claim 70, wherein theinsertion site is selected from the group consisting essentially of asurface location on the extein, a loop region of the extein, a proteasesensitive site within the extein, or at a position known to permitinsertion of one or more amino acid residues in the extein withoutinactivating the extein.
 76. The positive genetic selection system ofclaim 72, wherein the insertion site is selected from the groupconsisting essentially of a surface location on the extein, a loopregion of the extein, a protease sensitive site within the extein, or ata position known to permit insertion of one or more amino acid residuesin the extein without inactivating the extein.
 77. The reporter systemof claim 41 or 71, wherein the insertion site is selected from the groupconsisting essentially of a surface location on the extein, a loopregion of the extein, a protease sensitive site within the extein, or ata position known to permit insertion of one or more amino acid residuesin the extein without inactivating the extein.
 78. A positive geneticselection system for screening of agents which inhibit protein splicing,said selection system comprising: i) a host which contains a first geneencoding an inactivated form of a target enzyme which is required forcell viability under predetermined conditions; and ii) a second geneencoding an expressed form of said target enzyme, said second genecontaining an intein, wherein the inhibition of splicing of said targetenzyme by a given agent results in the reduced viability of said hostcell under said predetermined conditions wherein expression of saidtarget enzyme is required for viability or growth.
 79. A positiveselection system for the screening of temperatures which inhibit proteinsplicing in temperature sensitive inteins, said selection systemcomprising: i) a host cell which contains a first gene encoding aninactivated form of the target enzyme which is required for cellviability under predetermined conditions; and ii) a second gene encodingan expressed form of said target enzyme, said second gene containing anintein, wherein said intein is mutagenized and wherein the inhibition ofsplicing of said target enzyme at one or more predetermined temperaturesresults in the reduced viability of said host cell under saidpredetermined conditions wherein expression of said target enzyme isrequired for viability or growth.
 80. A method of identifying an agentfor antimicrobial activity against a microbial pathogen that naturallyhas an intein in an essential gene comprising screening for agents thatblock splicing of that intein in its native context or a homologousextein context.
 81. A method of identifying an agent for antimicrobialactivity against a microbial pathogen that naturally has an intein in anessential gene comprising screening for agents that block splicing ofthat intein in a heterologous extein context that includes one or morenative extein residues flanking one or both ends of the intein.
 82. Amethod of identifying agents with antimicrobial activity againstMycobacterium tuberculosis comprising screening for agents that inhibitsplicing of the Mycobacterium tuberculosis DnaB intein.
 83. A method ofidentifying agents with antimicrobial activity against Mycobacteriumleprae comprising screening for agents that inhibit splicing of theMycobacterium xenopi or Mycobacterium leprae GyrA inteins.
 84. A methodof identifying lead compounds with antimicrobial activity comprisingidentifying agents that inhibit splicing of an intein which is naturallypresent in that organism.